Uses of aloe products

ABSTRACT

Acemannan has been shown to be effective in treating a number of conditions where the principal mechanism of resolution or cure requires intervention by the patient&#39;s immune system. Acemannan has direct stimulatory effects on the immune system. Methods for treating cancer, viral diseases, respiratory and immune regulatory diseases, inflammations, infections and infestations by administering an acetylated mannan derivative, such as acemannan derived from aloe, are described. The method finds use in tissue cultures, animals and plants.

The present application is a divisional of U.S. application Ser. No.07/864,583, filed Apr. 7, 1992, now U.S. Pat. No. 5,308,838, which is adivisional of U.S. application Ser. No. 07/558,905, filed Jul. 27, 1990,now U.S. Pat. No. 5,118,673, which in turn, is a continuation-in-part ofU.S. application Ser. No. 07/229,164, filed Aug. 5, 1988, now U.S. Pat.No. 5,106,616 and entitled "Administration of Acemannan," the entirecontents and disclosure of which are hereby specifically incorporated byreference. Said U.S. application Ser. No. 07/229,164 corresponds toInternational Application PCT/US89/03381, filed Aug. 3, 1989, andpublished under International Publication No. WO 90/01253 on Feb. 22,1990, the entire contents and disclosure of which are also herebyspecifically incorporated by reference. The said U.S. application Ser.No. 07/229,164 is a continuation-in-part of U.S. application Ser. No.07/144,872, filed Jan. 14, 1988, and entitled "Process for Preparationof Aloe Products," granted on Jun. 25, 1989, as U.S. Pat. No. 4,851,224,the entire contents and disclosure of which are hereby specificallyincorporated by reference. Said U.S. Pat. No. 4,851,224 is acontinuation-in-part of U.S. application Ser. No. 06/869,261, filed onJun. 5, 1986, and entitled "Processes for Preparation of Aloe Products,Products Produced Thereby and Compositions Thereof," granted on Apr. 5,1988, as U.S. Pat. No. 4,735,935, the entire contents and disclosure ofwhich are also hereby specifically incorporated by reference. Said U.S.Pat. No. 4,735,935, corresponds to International Application No.PCT/US86/01335, filed Jun., 20, 1986, and published under InternationalPublication No. WO 87/00052 on Jan. 15, 1987, the entire contents anddisclosure of which are also hereby specifically incorporated byreference. Said U.S. Pat. No. 4,736,935 is a continuation-in-part ofU.S. application Ser. No. 06/810,025, filed Dec. 17, 1985 (nowabandoned), which is a continuation-in-part of U.S. application Ser. No.06/754,859, filed Jul. 12, 1985 (now abandoned) which is acontinuation-in-part of U.S. application Ser. No. 06/750,321 filed Jun.28, 1985 (now abandoned), which is a continuation-in-part of U.S.application Ser. No. 06/649,967 filed Sep. 12, 1984 (now abandoned),which is a continuation of U.S. application Ser. No. 06/375,720 filedMay 7, 1982 (now abandoned). Application Ser. No. 06/810,025 is entitled"Processes for Preparation of Aloe Products and Products ProducedThereby." Applications Ser. Nos. 06/754,959; 06/750,321; 06/659,967; and06/375,720 are entitled "Process for Preparation of Aloe Vera Products."

BACKGROUND OF THE INVENTION

A. Field of the Invention

The invention pertains to uses of biological response modifying agents.More particularly, this invention relates to the therapeutic use of apolysaccharide substance which is predominantly an acetylated mannan orits derivatives to:

1) relive the symptoms and/or treat the viral diseases of animals,including humans, other mammals, and birds, as well as of plants. Thesepolysaccharidic substances inhibit viral replication either alone, or incombination with other drugs, either through direct antiviral effects orthrough their immune stimulating activities;

2) enhance the response of the immune system to cancer in humans, othermammals, animals, birds and plants. These polysaccharidic substancesstimulate immune cells of the body and directly alter the tumor cellsurface so that the stimulated immune cells now recognize the tumorcells as "not self";

3) after the body's response to antigens, toxins, allergens and "self"antigens as seen in autoimmune diseases. These polysaccharidicsubstances cause immune regulator cells to function more appropriatelyto achieve homeostasis;

4) act as adjunctive therapy with other drugs in a wide range ofconditions where the final step in resolution or cure of the conditionrequires an immune response. These polysaccharidic substances can beused with anti-infective, antitumor, anti-inflammatory, andantidepressant drugs with no toxicity due to the polysaccharicicsubstance. The efficacy of the combination is superior over the singledrug alone.

B. Description of the General Background Information

Aloe is a member of the lily family. Hardin, Aloes of the World; AChecklist, Index and Code, Excelsa 9:57-94 (1979). Aloe barbadensisMiller is generally recognized as the "true aloe" because of its wideuse and, reportedly, most effective healing power, although in Japan,Aloe arborescens Miller traditionally has been used as a folk remedy forvarious ailments ranging from gastrointestinal disorders to athlete'sfoot. Aloe vera is a perennial plant with turgid green leaves joined atthe stem in a rosette pattern. The leaves of a mature plant may be morethan 25 inches long with sawlike spikes along their margins.

Aloe vera contains two major liquid sources, a yellow latex (exudate)and the clear gel (mucilage). The dried exudate of Aloe barbadensisMiller leaves is referred to as aloe. The commercial name is Curacaoaloe. It is composed mainly of aloin, aloe-emodin and phenols. Bruce,South African Medical Journal, 41:984 (1967); Morrow et al., Archives ofDermatology, 116:1064-1065 (1980; Mapp et al., Planta Medica, 18:361-365(1970); Rauwald, Archives Pharmazie, 315:477-478 (1982). A number ofphenolics, including anthraquinones and their glycosides, are known tobe pharmaceutically active, Bruce, Excelsa, 5:57-68 (1975); Suga et al.,Cosmetics and Toiletries, 98:105-108 (1983).

The mucilainous jelly from the parenchymal cells of the plant isreferred to as Aloe vera gel. There are generally no anthraquinones todecompose and cause discoloration of the gel unless the gel iscontaminated by an improper processing technique. Aloe vera gel is about98.5% water by weight. More than 60% of the total solid is made up ofpolysaccharides of carbohydrate origin. Organic acids and inorganiccompounds, especially calcium oxalate, account for the remainder of thesolid.

Whole leaves, exudates and fresh gels of Aloe plants have been used fora variety of human afflictions. Evidence of their use as a medicinalremedy can be traced to the Egyptians of 400 BC. Aloe vera was also usedto embalm the dead, as well as to protect the embalmers from thedeath-causing agent. Other early civilizations used Aloe vera for skincare, to relieve insect stings and bites, to treat scratches andulcerated skin, to promote wound healing, to prevent hair loss and as apurgative. It was the traditional medicine of many cultures as ananthelmintic, cathartic and stomachic and was used inter alia forleprosy, burns and allergic conditions. Cole et al., Archives ofDermatology and Syphilology, 47:250 (1943); Chopra et al., Glossary ofIndian Medicinal Plants, Council of Scientific and Industrial Research,New Delhi (1956); Ship, Journal of the American Medical Association,238(16):1770-1772 (1977); Morton, Atlas of Medicinal Plants of MiddleAmerican Bahamas to Yucatan, Charles C. Thomas Publisher, 78-80 (1981);Diez-Martinex, La Azbilia, Communicado NO. 46 Sobre Recursos BioticosPotenticiales del Pais, INIREB, Mexico (1981); Dastur, Medicinal Plantsof India and Pakistaon; D. B. Taraporevala Sons & Co., Private Ltd.,Bombay 16.17 (1962).

Aloe vera has enjoyed a long history of lay acceptance as possessing"curative" or "healing" qualities. Over the last few years, numerousbooks and articles meeting scientific standards have been written onAloe vera. Organizations such as the Aloe Vera Council and recognizedmedical institutions, through publications and case histories ofphysicians, veterinarians and other scientists, have given credence tothe "aloe phenomenon." Aloe vera has been featured extensively in thefield of dermatology, especially for treating radiation-caused skinconditions. Mackee, X-rays and Radium in the Treatment of Diseases ofthe Skin, 3rd Ed., Lea and Febiger, Phildelphia, 319-320 (1938); Rovattiet al., Industrial Medicine and Surgery, 28:364-368 (1959); Zawahry etal., Quotations From Medical Journals on Aloe Research, Ed. Max B.Skousen, Aloe Vera Research Institute, Cypress, Calif., 18-23 (1977);Cera et al., Journal of the American Animal Hospital Association,18:633-638 (1982). The body of scientific literature documenting medicalapplications in digestive problems, as a virucidal, bactericidal andfungicidal agent and in gynecological conditions is extensive and hasbeen adequately reviewed by Grindley et al., Journal ofEnthnopharmacology, 16:117-151 (1986)].

Depending on the way the leaves are processes, mucilage and sugars arethe major components of the dehydrated gel. The sugars found aregalactose, glucose, mannose, rhamnonse, xylose and uronic acids.Although reports conflict, the mucilage is mainly composed of mannan orglucomannan. Eberendu et al., The Chemical Characterization of Carisyn®(in preparation); Mandal et al., Carbohydrate Research, 86:247-257(1980b); Roboz et al., Journal of the American Chemical Society,70:3248-3249 (1948); Gowda et al., carbohydrate Research, 72:201-205(1979); Segal et al., Lloydia, 31:423 (1968).

Prior to this work, the controversy over the identity of the activesubstance(s) in Aloe vera had not been settled. It is thereforeimportant to clearly distinguish between the components present in thegel and those found in the exudates. A majority of the gel is a mucilageof mainly polysaccharide nature with minor amounts of various othercompounds. It has been observed that in some of the activities there maybe some synergistic action between the polysaccharide base and othercomponents. Leung, Excelsa 8:65-68 (1978); Henry, Cosmetics andToiletries, 94:42-43, 46, 48, 50 (1979). For example, several workersreport that the effective components for wound healing may be tannicacid [Freytag, Pharmazie, 9:705 (1954)] and a kind of polysaccharide.Kameyama, Wound-healing compositions from Aloe arborescens extracts.Japanese Patent #7856995, (1979). Mackee, supra, noted that the gel, notthe rind or the exudate, was responsible for the beneficial effects inthe treatment of radiation burns, and he stressed the importance ofusing fresh leaves for effective treatment. Polysaccharides degrade withtime, and certain molecular weight sizes may be necessary to elicit aspecific pharmacological response. Goto et al., Gann, 63:371-374 (1972).

However, there are many examples in the literature indicating thatpolysaccharides can exhibit pharmacological and physiological activitieswithout help from other components. Gialdroni-Grassi, InternationalArchives of Allergy and Applied Immunology, 76(Supp. 1):119-127 (1985);Ohno et al., Chemical and Pharmaceutical Bulletin, 33(6):2564-2568(1985); Leibovici et al., Chemico-Biological Interactions, 60:191-200(1986); Ukai et al., Chemical and Pharmaceutical Bellutin, 31:741-744(1983); Leibovici et al., Anticancer Research, 5:553-448 (1985). Onesuch example relates to development of atherosclerosis. Hyperlipidemiais the general population and especially in familialhypercholesterolemia is associated with coronary heart disease anddeath. In countries where dietary fiber intake is high, atherosclerosisappear to be uncommon. Trowell et al., Editors, Refined CarbohydrateFoods and Disease, London, Academic Press, 207 (1975). Pectin and guarare reported to lower cholesterol in normal hyperlipidemic patients. Kayet al., American Journal of Clinical Nutrition, 30:171-175 (1977).Locust beam gum, a polysaccharide composed of mannose and galactose,decreased the plasma lipoprotein cholesterol concentrations in bothnormal and familial hypercholesterolemic subjects. Zavoral et al.,American Journal of Clinical Nutrition, 38:285-294 (1983). Addition ofguar gum to carbohydrate meals decreased the postprandial rise ofglucose in both normal and diabetic subjects. Jenkins et al., Lancet,2:779-780 (1977). Kuhl et al., in Diabetes Care, 6(2):152-154 (1983)demonstrated that guar gum exhibited glycemic control of pregnantinsulin-dependent diabetic patients.

The antitumor activity of polysaccharides has been widely reported.Polysaccharides prepared from Lentinus cyathiformis are known toincrease host defense against tumors. Rethy et al., Annales ImmunologiaeHungaricae, 21:285-290 (1981). There are several reports thatpolysaccharides from mushroom, yeast or bacterial extracts can elicit ahigh degree of host defense activity against viral and tumorinfestations. Chihara, Nature, 222:687 (1969); Shwartzman et al.,Proceedings of the Society for Experimental Biology and Medicine,29:737-741 (1932); Suzuki et al., Journal of Pharmacobio-Dynamics,7(7):492-500 (1984), also reported antitumor activity of apolysaccharide fraction (GF-1) extracted from cultured fruiting bodiesof a fungus, Grifola frondosa. This fraction showed equivalent, highlevels of inhibiting activity when administered intraperitoneally (IP),intravenously (IV) and intratumorally (IT). However, oral administration(PO) was not effective. The GF-1 fraction also exhibited antitumoraction against the solid form of Meth A fibrosarcoma and MM 46 carcinomain mice. Lentinan, which is a 6-branched β-1-3-liked glucan similar toGR-1, was ineffective against Mech A fibrosarcoma. Chihara, "Theantitumor polysaccharide Lentinan: an overview;" Manipulatoin of HostDefense Mechanisms; Ed. by Aoki et al., Excerpta Medica, North Holland,1-16 (1981); Sasaki et al., Carbohydrate Research, 47(1):99-104 (1976).Synthesized branched polysaccharides were reported to demonstrateactivities against tumors. Matsuzaki et al., Makromol. Chem.,186(3):449-456 (1985). Matsuzaki et al. [Makromol. Chem., 187(2):325-331(1986)] synthesized branched polysaccharides, which showed significantactivities, from ivory nut mannan (β-(1-4)-D-mannopyranose) andβ-(1-4)-linked glucomannan. A partially acetylated linearβ-(1-3)-D-mannan extracted from fruit bodies of Dictyophoria indusiataFisch, also exhibited antitumor activity. Hara, Carbohydrate Research,143:111 (1982). It appears that antitumor action depends on the type ofpolymer main chain and its degree of polymerization, becauseβ-(1-3)-glucan-type polymers show higher antitumor activity thanβ-(1-4)-glucan and hemicellulosic polymers. Matsuzaki et al., Makromol,Chem., 187:325-331 (1986). A carboxymethylated derivative ofβ-(1-3)-glucan obtained from bacterial culture filtrate caused severecell loss from established sarcoma 180 tumors within 2 hours after theinjection of the derivative. Baba, Journal of Immunopharmacology, 8(6):569-572 (1986). The same author observed a compensatory increase inpolymorphonuclear leukocytes due to injection of the substance.Incidentally, bestatin, a dipeptide known to possess immune-modulatingand antitumor activity [Ishizuka, Journal of Antibiotics, 32:642-652(1980)], influenced neither the tumor yield nor the polymorphonuclearleukocyte count. Baba et al., supra.

There are numerous reports on the antitumor effect of sulfatedpolysaccharides, including heparin [Jolles et al., Acta Univ. Int.Cancer, 16:682-685 (1960); Suemasu et al., Gann, 61(2):125-130 (1970)],sulfated laminaran and dextran [Jolles et al., British Journal ofCancer, 17:109-115 (1963)]. Yamamoto et al., in Japanese Journal ofExperimental Medicine, 54:143-151 (1984), reported enhancement ofantitumor activity of a fucoidan fraction by further sulfation. Thesulfated product demonstrated activity against L-1210 leukemia. Theauthors postulated that the mechanism of the antitumor action might bedue partly to inhibition of invasive growth of L-1210 cells, as a resultof electrostatic repulsion between the tumor cell and mesothelial cells.Yamamoto et al., supra. Polysaccharides with sulfate groups are alsoreported to be human T cell mitogens and murine polyclonal B cellactivators. Sugawara et al., Microbiological Immunology, 28(7):831-839(1984). Generally, homopolysaccharides of high molecular weight withsulfate groups possess these properties. Dorries, European Journal ofImmunology, 4:230-233 (1974); Sugawara et al., Cell Immunology,74:162-171 (1982).

It has been reported that glucan extracted from the yeast Saccharamycescervisiae is a modulator of cellular and humoral immunity. Wooles etal., Science, 142:1078-1080 (1963). The polysaccharide also stimulatedproliferation of murine pluripotent hematopoietic stem cells,granulocyte macrophage colony-forming cells and cells forming myeloidand erythroid colonies. Pospisil et al., Experientia, 38:1232-1234(1982); Burgaleta, Cancer Research, 37:1739-1742 (1977). Maisin et al.,[Radiation Research, 105:276-281 (1986)] also reported that IVadministration of a polysaccharide induced protection of murinehematopoietic stem cells against x-ray exposure, thereby decreasing themortality of the mice so exposed.

Lackovic et al., [Proceedings of the Society for Experimental Biologyand Medicine, 134:874-879. (1970)], took yeast cell-wall and extractedall constituent matter leaving only "mannans" that he found to beresponsible for the induction of α-interferon production by monocytes.The "purified mannans" alleged to be responsible for the physiologicresponse had a molecular weight of 5,500-20,000 daltons. He theorizedthat mannans stimulated mouse peritoneal macrophages to produce theλ-interferon. He also stated that the mannans he isolated showed notoxicity and "they are poor antigens." There was no mention by Lackovicet al., of the use of these "purified mannans" for antiviral activity orfor IL-1 stimulation. We submit that Lackovic et al.'s "purifiedmannans" comprised an assortment of unknown and unidentified substitutedand unsubstituted mannans.

Seljelid et al., [Experimental Cell Research, 131(1):121-129 (1981)]have observed that insoluble or gel-forming glycans activatedmacrophages in vitro, whereas the corresponding soluble glycans did not.They postulated that the orientation in which the glycan was presentedto the mononuclear phagocyte was decisive for activation. Bogwald,[Scandinavian Journal of Immunology, 20:355-360 (1984)] immobilizedglycans that had a stimulatory effect on the macrophages in vitro. Thisled the authors to believe that the spatial arrangement of the glycanwas decisive for the effect on the macrophages in vitro. A purifiedpolysaccharide isolated from Candida albicans induced an antibodyresponse by human peripheral blood lymphocytes in vitro. Wirz et al.,Clinical Immunology and Immunopathology, 33:199-209 (1984). There weresignificant differences between the anti-Candida antibodies in sera ofnormal and Candida-infected individuals. Wirz et al., supra.

The antiviral activity of polysaccharides and polysaccharides linked topeptides has been observed. Suzuki et al., Journal of Antibodies,32:1336-1345 (1979). Suzuki et al,., supra, reported an antiviral actionof peptidomannan (KS-2) extracted from mycelial culture of Lentinusedodes. Both oral and intraperitoneal administration increased the peakserum interferon titer, which protected mice against viral infections.This was different from dextran phosphate (DP-40) [Suzuki et al.,Proceedings of the Society for Experimental Biology and Medicine,149(4):1069-1075 (1975)] and 9-methylstreptimidone (9-MS) [Saito et al.,Antimier. Agent & Chemotherapy, 10(1):14-19 (1976)], which inducedhigher titers of interferon in mice only if administered IV or IP.

Anti-inflammatory activity of Aloe vera gel has been widely reported byboth oral testimonies and respected scientific journals. Rubel[Cosmetics and Toiletries, 98:109-114 (1983)], discussed fully thepossible mechanism of the anti-inflammatory effect of aloe gel. Ukai etal., [Journal of Pharmacobio-Dynamics, 6(12):983-990 (1983)] notedanti-inflammatory activity of polysaccharides extracted from thefruiting bodies of several fungi. The polysaccharides demonstrated asignificant inhibitory effect on carrageenan-induced edema. One of thepolymers, O-acetylated-D-mannan (T-2-HN), in addition demonstrated amore marked inhibitory effect than phenylbutazone on scald hyperalgesia.Ukai et al., supra. The assertion that the polysaccharide is free fromprotein and lipids strongly suggests that the anti-inflammatory effectis due to the acetylated mannan only.

Other researchers have also reported anti-inflammatory effects ofcomplex polysaccharides [Saeki et al., Japanese Journal of Pharmacology,24(1):109-118 (1974)], glycoproteins [Arita et al., Journal ofBiochemistry, 76(4):861-869 (1974)] and sulfated polysaccharides [Rochaet al., Biochemical Pharmacology, 18:1285-1295 (1969)].

Literature which reports that polysaccharides possess pharmacologicaland physiological activities continues to flood the pages ofwell-respected scientific journals. It is therefore logical that themucilaginous gel of the Aloe vera plant, which is essentially apolysaccharide, holds the secret to Aloe vera's medicinal properties.The controversy over whether the polysaccharide is a glucomannan,mannan, pectin, or of some other composition, is resolved by a series ofchemical purification steps. Yagi et al., [Planta Medica, 31(1):17-20(1977)], using a slightly modified extraction method; isolatedacetylated mannan (aloe mannan) from Aloe arborescens Miller var.natalensis. Ovodova [Khim. Prior. Soedin, 11(1):325-331 (1975)],however, earlier isolated pectin as the main component of the same aloespecies. As discussed above, the biological activity of polysaccharideshas been recognized for many years. Polysaccharide materials recoveredfrom plants, yeast and bacteria have demonstrated direct biologicalactivity by eliciting an increase in host defense systems. This reactionis primarily manifested by increased host surveillance for otherantigenic substances. Polysacchardies serve as adjuvants (DEAE Dextran,etc.) and immunomodulators. They also can function as unique Tcell-independent antigens. Both cellular and humoral immunity may beaffected, and increased phagocytosis of infectious organisms and tumorcells has been observed, as has enhanced production of immunoglobulins.

The structure of these immunologically active polysacchardies and thetypes of structural variations appear to be the factors that controltheir potency and toxicity. Their mode(s) of action remain poorlyunderstood; however, recent evidence indicates that severalpolysaccharides induce lymphocytes and macrophages to produce a widerange of immunologically active substances. For example,2-keto-3-deoxy-D-manno-octulosonic acid (KDO) appears to be the chemicalportion of lipopolysaccharide (LPS) that provides the minimum signal formacrophage host defense activation [Lebbar et al., Eur. J. Immunol.16(1):87-91 (1986)]. The composition of the present invention possessesall of the attributes of these immunologically active substances; it isamong the most potent of all known biologically active polysaccharidesbut differs in that no toxicity has been observed. It also manifestsspecific antiviral activity through alteration of viral glycoproteinsynthesis.

A number of pharmacology studies have been conducted on Aloe vera gel inrecent times. Results have included more rapid healing of radiationburns [Rowe, J. Am. Pharm. Assoc., 29:348-350 (1940)] and acceleratedhealing of wounds [Lushbaugh et al., Cancer, 6:690-698 (1953)]. Thermalburns treated with Aloe vera gel heal much faster than untreated burns[Ashley et al., Plast. Reconstr. Surg., 20:383-396 (1957), Rovatto,supra, Rodriguez-Bigas et al., J. Plast. Reconstr. Surg., 81:386-389(1988)]. The gel is useful in treating leg ulcers [El Zawahry et al.,Int. J. Dermatol., 12:68-73 (1973)] and in hastening post surgicalhealing (Payne, Thesis submitted to Faculty of Baylor University, Waco,Tex. MS Degree). Experimental evidence suggests that extracts of Aloevera have anti-infectious properties [Solar, Arch. Inst. PasteurMadagascar, 47:9-39 (1979)] and enhance phagocytosis [Stepanova, Fiziol.Akt. Veshchestva, 9:94-97 (1977)].

The active fraction of Aloe vera gel has been identified by CarringtonLaboratories, Inc., Irving, Tex., as a long-chain polyolisperseβ-(1,4)-linked acetylated mannan interspersed with O-acetyl groupshaving a mannose monomer-to-acetyl group ratio of approximately 1:0.91.Acemannan is the nonproprietary name of the biologically activecomponent of Carrisyn®, a compound isolated and developed by CarringtonLaboratories, Inc. See U.S. Pat. No. 4,735,935, U.S. Pat. No. 4,851,224,and the U.S. patent application Ser. No. 07/229,164, and referencescited therein, the disclosures of all of which are incorporated hereinby reference. All of these patents and this patent application are alsoassigned to Carrington Laboratories, Inc.

Mannans, including glucomannans and galactomannans, have long been usedby man. For example, galactomannans, in the form of plant gums, arewidely employed as binders for control of food texture. In addition,some mannans have exhibited significant therapeutic properties (Davisand Lewis, eds. Jeanes A., Hodge J., In: American Chemical SocietySymposium, Series 15. Washington, D.C., American Chemical Society,1975). Practitioners of Japanese folk medicine have long believed thatextracts of certain fungi have anticancer activity. On investigation,many of these extracts have been found to contain complex carbohydrateswith immune-stimulating activity. These carbohydrates are usuallypolymers of mannose (mannans), glucose (glucans), xylose(hemicellulose(, fructose (levans) and mixtures of these. Individualsugars may be bonded in different ways and chains may be branched orunbranched. Glucans have ben the most widely studies of theseimmunostimulatory carbohydrates. It has become increasingly clear thateven though they have no toxicity mannans are as effective, if not moreeffective, than glucans.

Pure mannans are relatively uncommon in higher plants, although they area major structural component of some yeasts. For example, about 45% ofthe cell wall of Sccharomyces cerevisiae consists of a mannan. Thismannan is a water soluble molecule composed of β-(1,6)-, β-(1,3)-, andβ-(1,2)-linked, partially phosphorylated D-mannose residues [McMurroughetr al., Biochem. J., 105:189-203 (1967)]. Other biologically activemannans have been obtained from Candida utilis [Oka et al., Gann,60:287-293 (1969(, Oka et al., Gann, 58:35-42 (1968)], Candida albicans,Coccidioides immitis and Rhodotorulum rubrum [Wheat et al., Infect.Immun., 41:728-734, (1983)]. Mannans (including galactomannans andglucomannans) are relatively resistant to attack by mannosidases but canbe degraded by exo- and endo-mannanases [Emi, et al., Agr. Biol. Chem.,36:991-1001 (1972), Snaith, et al., Adv. Carbohydr. Chem. Biochem.,28:401-445, (1973) Herman, Am. J. Clin. Nutr., 24:488-498 (1971),McMaster, et al., Proc. Soc. Exp. Biol. Med., 135:87-90 (1970), Jones etal., J. Biol. Chem. 243:2442-2446 (1968), Eriksson et al., Acta. Chem.Scand., 22:1924-1934 (1968)]. The most marked biological activities ofmannans in mammals are activation of macrophages and stimulation of Tcells. As a result, they are potent immunostimulants with significantactivity against infectious diseases and tumors [Hasenclever et al., J.Immun. 93:763-771 (1964)].

Saccharamyces mannan (15 mg/kg/day) enhances carbon clearance in normalmale ddI mice, presumably acting as a reticuloendothelial systemstimulant [Suzuki et al., Gann, 62:553-556 (1971)]. This same mannanalso increases the number of antibody-forming cells in the spleen[Suzuki et al., Gann, 62:343-352 (1971)]. In vitro studies with mouseperitoneal cells (a mixture of macrophages and lymphocytes) indicatethat some mannans and mannan-protein complexes can stimulate interferonrelease both in vivo and in vitro [lackovic et al., Proc. Soc. Exp.Biol. Med., 134:874-879 (1970)]. The mannans stimulated interferonrelease in a manner similar to endotoxins but, in contrast toendotoxins, caused minimal toxicity (Borecky et al., Acta Virol.,11:264-266 (1967), Hasenclever, supra). The mannan from Candida albicansis active in this way, but the mannan from Saccharomyces cerevisiae isinactive [DeClercq et al., Ann. NY Acad. Sci., 173:444-461 (1970)].Inconsistent or poor results have been obtained in other laboratories(DeClercq, supra). These differences may be due to slight structural orsize differences in the polymers [Suzuki et al., Jpn. J. Microbiol.,12:19-24 (1968)]. The latter is more likely responsible since lowmolecular weight mannans (5.5-20 kDa) tend to be most active in theinterferon-inducing assay, also Saccharomyces mannan tends to be largerthan Candida mannan.

A galactomannan of 20 kDa from Lipomyces starkeyi had weakinterferon-inducing properties. In contrast, Candida albicans mannaninduced the appearance of interferon activity 2-24 hrs after intravenousadministration (Borecky, supra).

DMG, a degraded mannoglucan from Microellobosporia grisea culture fluid,can stimulate cytotoxic activities of macrophages, natural killer (NK)cells and killer T cells, and it enhances the secretion of interleukin-1(IL-1) and colony-stimulating factors (CSF). It has more potentantitumor activity than lentinan (a glucan from Lentinus edodes)[Nakajima et al., Gann, 75:260-268, (1984), Inoue et al., Carbohyd.Res., 114:164-168 (1983)]. DMG stimulates macrophages to produceincreased amounts of IL-1. In addition, DMG enhances 1) antibodyproduction against sheep erythrocytes, 2) natural killer activity ofspleen as well as of peritoneal cells, and 3) cytostatic activity ofperitoneal macrophages [Nakajima et al., Gann, 75:253-259 (1984)].

Mannose-binding proteins have ben identified in the serum of rabbits andin the liver of humans and laboratory rodents. These proteins can bindglucomannans such as those found in cell walls of bacteria, yeasts,fungi and in envelope glycoproteins of certain viruses such as the humanimmunodeficiency virus (HIV). In humans, the major mannose-bindingprotein is an acute-phase protein; its levels rise in stressedindividuals [Ezekowitz et al., J. Exp. Med., 169:185-196 (1989)]. Theenvelope glycoproteins of the human immunodeficiency virus (HIV gp120and gp41) contain mannose-rich oligosaccharides that appear to bepotential ligands for the mannose-binding protein. As a result, themannose-binding protein can inhibit HIV infection of lymphoblasts andbind selectively to HIV-infected cells. Free yeast mannan cancompetitively interfere with binding of this protein to infected cells.Thus, factors that induce an increase in the level of themannose-binding protein may confer protection against HIV.

Problems to Which The Invention is Addressed

Virus, cancer and diseases of immune regulation continue to be majorcauses of both morbidity and mortality in humans, other mammals, otheranimals, birds, and plants. Problems associated with currently useddrugs are, namely, general toxicity, lack of efficacy (or both),deficiency in specificity and development of resistance by causativeorganisms or agents. Hence, better non-toxic yet therapeuticallyefficient agents are needed for the treatment of these diseases.Acemannan has been shown to possess a unique combination ofimmunodulatory and antiviral properties.

SUMMARY OF THE INVENTION

It is therefore an object to provide a method of enhancing orstimulating the immune system in an animal, comprising theadministration of an amount of acetylated mannan derivative sufficientto effect the enhancement and the stimulation of the immune system inthe animal.

It is also an object to provide a method of activating, inducing, and/orenhancing in an animal the synthesis and production of cytokines (suchas interleukins, interferon, and prostaglandin) by monocytes andmacrophages, peripheral blood adherent cells, comprising theadministration of an amount of an acetylated mannan derivative to theanimal sufficient to effect monocyte and macrophage activation.

It is a further object to provide a method of stimulating macrophagephagocytosis in an animal, comprising the administration of an amount ofan acetylated mannan derivative sufficient to effect monocyte andmacrophage activation.

It is still a further object to provide a method of producing anantiviral effect in a tissue culture, animal, or plant, comprising theadministration of a sufficient amount of an acetylated mannan derivativeinto the tissue culture, animal, or plant to produce the antiviraleffect.

It is still a further object to provide a method of producing defectivevirus in a human infected with virus, comprising the administration ofan amount of an acetylated mannan derivative into the human sufficientto effect monocyte and macrophage activation and alter viral replicationin cells infected with virus.

It is another object to provide a method of producing an antiviraleffect in an animal, comprising the administration of an amount of anacetylated mannan derivative into the animal sufficient to induceinterferon synthesis, enhance antibody formation, enhance T-cellactivities, enhance killer cell activities, stimulate thymic activity,alter glycosylation of glycoprotein, alter second messenger synthesisand activity, inhibit viral replication, or a combination of any of theabove.

It is still a further object to provide a method of producing defectivevirus in a master seed culture for vaccine production, comprising addinga predetermined amount of an acetylated mannan derivative into themaster seed culture sufficient to produce altered viral replication.

It is still a further object to provide a method of stimulating andenhancing cytokine synthesis by cells of the immune system, comprisingthe administration of an amount of an acetylated mannan derivative intothe animal sufficient to stimulate cytokine synthesis.

It is yet another object of the present invention to provide a method ofinducing the immune system of a plant or an animal to inhibit the growthof a tumor or a cancer, comprising the administration of a sufficientamount of an acetylated mannan derivative into the plant or the animalto cause the immune system of the plant or animal to inhibit the growthof a tumor or a cancer.

It is also an object of the present invention to provide a method ofcausing the immune system of a plant or an animal to destroy or inhibitthe growth of a tumor or a cancer, comprising the administration of asufficient amount of an acetylated mannan derivative into the plant oranimal to cause the immune system of the plant or animal to recognizethe tumor or cancer as "not self."

It is a further object of the present invention to provide a method ofproducing anticancer effects in animals that have succumbed to cancer ofviral, chemical, radiation, genetic or other origins.

It is still a further object to provide a method of producing anantitumor effect in an animal that has succumbed to tumors of geneticorigins, comprising the administration of an amount of an acetylatedmannan derivative into the animal sufficient to inhibit primary andsecondary messenger expression of oncogenes.

It is yet another object to provide a method of reducing tissue damage,such as ulceration and/or necrosis, and of restoring soft-tissuecapillary bed vascular perfusion in an animal, comprising theadministration of an amount of an acetylated mannan derivative into theanimal sufficient to restore tissue viability.

It is also an object to provide a method of reducing the symptomsassociated with inflammatory bowel diseases in an animal, comprising theadministration of an amount of an acetylated mannan derivative into theanimal sufficient to reduce the symptoms associated with inflammatorybowel disease.

It is further an object to provide a method of reducing symptomsassociated with multiple sclerosis in a human, comprising theadministration of an amount of acetylated mannan derivative into thehuman sufficient to reduce symptoms associated with multiple sclerosis.

It is also an object to provide a method of reducing the symptomsassociated with neurochemical disorders and depression in an animal,comprising the administration of an amount of acetylated mannanderivative into the animal sufficient to reduce the symptoms associatedwith neurochemical disorders and depression.

It is a further object to provide a method of treatment of acute andchronic autoimmune disease in an animal, comprising the administrationof an acetylated mannan derivative into the animal sufficient to causeimmunosuppression and/or immunomodulation of the cells and tissuesresponsible for the automimmune disease.

It is still a further object to provide a method of causing a more rapidhealing of traumatic injuries in an animal, comprising theadministration of an acetylated mannan derivative into the animalsufficient to cause the animal's body tissue repair mechanism and immunesystem to respond more rapidly and appropriately to a trauma.

It is a further object to provide a method of causing an affect on therespiratory system of an animal to ameliorate the symptoms associatedwith asthma, conjunctivitis, rhinitis and bronchitis, comprising theadministration of an acetylated mannan derivative into the animalsufficient to cause immunomodulation of the cells and tissuesresponsible for the symptoms associated with asthma, conjunctivitis,rhinitis and bronchitis.

It is still a further object to provide a method of producing aprophylactic effect in an animal resulting in the prevention ofinfection by infectious organisms, comprising administration of anacetylated mannan derivative into the animal sufficient to cause theanimal body's immune system to prevent infection by an infectiousorganism.

It is also an object to provide a method of reactivating enzyme systemsand organ systems to cause a return to function of age-depleted tissue,comprising the administration of an acetylated mannan derivative intothe animal sufficient to cause the animal body and its tissue to producecell products and up-regulate genes which cause the tissue to return tofunction and express juvenile cell function and characteristics.

It is a still further object to provide a method of immunoenhancingvaccines by the production of an adjuvant effect, comprising adding apredetermined amount of an acetylated mannan derivative into the vaccineproduct.

It is a still further object to provide a method of treating an animalafflicted with a tumor, comprising administration to the animal anamount of an acetylated mannan derivative sufficient to effect monocyteand macrophage activation and enhance natural killer cell activity andspecific tumor cell lysis by cytotoxic cells and/or antibodies.

It is a still further object to provide a method of introducing anacetylated mannan derivative into the cellular organelles of (i) anoninfected cell to give rise to altered glycoproteins which providesaid cell with protection from viral infection and/or of (ii) avirus-infected cell to produce glycoproteins which destroy or inhibitviral expression in said infected cell, comprising introducing asufficient amount of an acetylated mannan derivative into the cell toalter viral glycoproteins in or at the surface of the cell.

It is still a further object to provide a method of introducing anacetylated mannan derivative into the cellular organelles of avirus-infected cell to produce glycoproteins which prevent or inhibitviral expression in said infected cell wherein the acetylated mannanderivative is introduced into the cell in an amount sufficient to renderthe virus noninfective.

It is a still further object to provide a method of introducing anacetylated mannan derivative into the cellular organelles of avirus-infected cell to produce altered glycoproteins which prevent orinhibit viral expression in said infected cell wherein the cell isvirus-infected, comprising the administration of the acetylated mannanderivative into the cell in an amount sufficient to (i) to cause a broadspectrum of specific antibodies to be produced which provide a broaderimmunological response than the cell had prior to introduction, and (ii)to enhance the rate of broad spectrum antibody production.

It is also an object of this invention to provide a method ofincreasing, in an animal, amounts of acetylated mannan derivative tointra- and extra-cellular metabolic pathways to correct malabsorptionand mucosal cell maturation syndromes in an animal, comprising the stepof administration to the animal an amount of the acetylated mannanderivative sufficient to provide additional acetylated mannan derivativefor the synthesis of glycoprotein thus accelerating Michaelis-Menten(K_(m)) kinetics for mannosyl transferase activity.

It is a further object to provide a method of inducing a virus-infectedmammalian cell to express altered viral glycoprotein antigens on itssurface which will initiate an antibody-dependent cell cytolysis (ADCC)by cytotoxic lymphocytes, comprising administration to the mammal anamount of an acetylated mannan derivative into the infected cellsufficient to produce altered viral glycoproteins and to cause thealtered viral glycoproteins to be expressed on the surface of theinfected cells and thus expose them to humoral antibodies.

It is still another object to provide a method of introducing anacetylated mannan derivative into a human to reduce the symptomsassociated with multiple sclerosis, comprising administration to thehuman an amount of the acetylated mannan derivative sufficient to reduceplaque formation and to induce plaque replacement with functional tissuein the central nervous system cells.

It is also an object to provide a method of introducing an acetylatedmannan derivative into a mammal to reduce the symptoms associated withinflammatory bowel disease, comprising the administration to the mammalan amount of the acetylated mannan derivative sufficient to resolvelesions associated with inflammatory bowel disease by increasing tissueregeneration of ulcers in said lesions and by reducing autoimmuneimmunoglobulin in local tissues of said lesions.

DESCRIPTION OF THE FIGURES

FIG. 1 shows synergistic antiviral effects of acemannan and AZT on theviability of HIV-infected MT-2 cells.

FIG. 2 shows synergistic antiviral effects of acemannan and AZT asquantified by the percent increase in viability of HIV-infected MT-2cells.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Carrisyn® is the brand name given by the assignee of the instantinvention to the purified ethyl alcohol extract of the inner gel of theleaves of Aloe barbadensis Miller. The active component of Carrisyn® hasbeen designated "acemannan" by the United States Adopted Name Council.Not less than 73% of Carrisyn® extract is acemannan; Carrisyn® extractcomprises generally about 73% to 90% acemannan. Carrisyn® extract isgenerally produced by removing the outer sheath of the leaf, thenremoving and processing the inner filet or mucilage as follows: pHadjustment, ethanol extraction, freeze drying and grinding. See U.S.application Ser. No. 144,872 filed January 1988, a continuation-in-partof U.S. application Ser. No. 869,261 (now U.S. Pat. No. 4,735,935), thedisclosures of all of which are incorporated herein by reference.Processing in this manner predicts that essentially no covalent bondsare altered and therefore no toxic compounds are created. Thesemanufacturing steps were developed to overcome the inability oftraditional aloe product producers to standardize and stabilize thepolysaccharides.

Carrisyn is a fluffy, white, amorphous powder, which is poorly solublein water and dimethyl sulfoxide and insoluble in most other organicsolvents. This powder contains not less than 73% of a polysaccharideconsisting essentially of linear β(1-4)-D-mannosyl units. Thepolysaccharide is a long chain polymer interspersed randomly with acetylgroups linked to the polymer through an oxygen atom. The generic namefor the polymer is acemannan. The degree of acetylation is approximately0.91 acetyl groups per monomer as determined by the alkaline hydroxamatemethod. See Hestrin, Journal of Biological Chemistry, 180:240-261(1949). Neutral sugars linkage analysis indicates that attached to thechain, probably through an α(1-6) linkage, is a D-galactopyranose in theratio of approximately one for every 70 sugars. The 20:1 ratio ofmannose to galactose indicates that galactose units are also linkedtogether, primarily by a β(1-4) glycosidic bond. The chemical structureof acemannan may be represented as follows: ##STR1##

Definition of Terms

The term "virus" as used herein includes both the DNA and the RNA virus.It can either be an enveloped or a non-enveloped virus. The term"enveloped virus" in all cases but one is understood to mean a virusencased within a modified host cell membrane; the poxviruses producetheir own envelope. Typical enveloped viruses are set forth in Table 1.

                  TABLE 1                                                         ______________________________________                                        The following are enveloped viruses as divided into family                    and common species or genus:                                                  Family       Common Species or Genus                                          ______________________________________                                        Herpesviridae                                                                              human herpes simplex virus types I & II                                       bovine mammillitis virus                                                      herpes B virus of monkeys                                                     pseudorabies virus                                                            equine rhinopneumonitis virus                                                 varicella-zoster virus                                                        human cytomegaloviruses                                                       murine cytomegaloviruses                                                      Epstein-Barr virus                                                            Baboon herpes virus                                                           Chimpanzee herpes virus                                                       Marek's disease herpes virus                                                  Hinze virus                                                                   Turkey herpes virus                                                           Herpes virus ateles                                                           Herpes virus saimiri                                                          Infectious bovine rhinotracheitis virus                          Iridoviridae African swine fever virus                                                     Frog virus group (Ranavirus)                                                  Iridovirus                                                                    Chloriridovirus                                                  Poxviridae   vaccinia virus                                                                smallpox virus                                                                cowpox virus                                                                  monkeypox virus                                                               buffalopox virus                                                              camelpox virus                                                                ectromelia of mice virus                                                      rabbitpox virus                                                               Orf virus                                                                     avipox virus                                                                  sheep-pox virus                                                               goatpox virus                                                                 lumpy skin disease (Neethling) virus                                          myxoma virus of hares                                                         fibroma viruses of rabbits                                                    fibroma viruses of squirrels                                                  swinepox virus                                                                Yaba monkey virus                                                             molluscum contagiosum virus                                      Hepadnaviridae                                                                             human hepatitis B virus (HBV)                                                 woodchuck hepatitis virus                                                     ground squirrel hepatitis virus                                               duck hepatitis virus                                             Orthomyxoviridae                                                                           Influenza virus, types A, B, and C                               Paramyxoviridae                                                                            Newcastle disease virus of fowl                                               human parainfluenza viruses                                                   Sendai virus                                                                  mumps virus                                                                   paramyxoviruses                                                               measles virus                                                                 rinderpest virus of cattle                                                    canine distemper virus                                                        peste-des-petits-ruminants virus                                              of sheep and goats                                                            respiratory syncytial virus of man                                            bovine respiratory syncytial virus                                            pneumonia virus of mice                                          Rhabdoviridae                                                                              rabies virus                                                                  vesicular stomatitis virus of:                                                horses, cattle and swine                                                      chandipura virus                                                              lyssavirus                                                                    duvenhage virus                                                               Lagos bat virus                                                               mokola virus                                                     Bunyaviridae bunyavirus (Bunyamwera, Bwamba,                                               California, Capim, Guama, phlebovirus                                         koongol, patois, simbu and tete viruses)                                      sandfly fever virus                                                           Rift Valley fever virus of sheep and                                          ruminants                                                                     Nairovirus                                                                    Crimean-Congo                                                                 Hemorrhagic fever viruses                                                     Uukuvirus                                                                     Uukuniemi virus                                                               Hantaan virus                                                                 Korean hemorrhagic fever virus                                   Filoviridae  ebola virus                                                                   Marburg virus                                                    Nodaviridae  Nodamura virus                                                   Togaviridae  Alphaviruses                                                                  aura virus                                                                    Chikungunya virus                                                             eastern equine encephalitis virus                                             getah virus                                                                   mayaro virus                                                                  middleburg virus                                                              mucamba virus                                                                 ndumu virus                                                                   O'Nyong-nyong virus                                                           pixuna virus                                                                  ross river virus                                                              semliki forest virus                                                          sindbis virus                                                                 una virus                                                                     Venezuelan equine encephalitis virus                                          western equine encephalitis virus                                             Whataroa virus                                                                rubella virus                                                                 mucosal disease virus                                                         border disease virus                                                          hog cholera virus                                                Flaviviridae flavivirus                                                                    Brazilian encephalitis virus                                                  Bussuquara virus                                                              dengue virus                                                                  iiheus virus                                                                  Israel turkey meningoencephalitis virus                                       Japanese B encephalitis virus                                                 kunjin virus                                                                  Kyasanur forest disease virus                                                 langat virus                                                                  louping ill virus                                                             modoc virus                                                                   Murray valley encephalitis virus                                              ntaya virus                                                                   omsk hemorrhagic fever virus                                                  powassan virus                                                                St. Louis encephalitis virus                                                  spondwnei virus                                                               tick-borne encephalitis                                                       Uganda S virus                                                                US bat salivary gland virus                                                   wesselsbron virus                                                             west nile fever virus                                                         yellow fever virus                                                            zika virus                                                                    European tick-borne encephalitis                                              Far Eastem tick-borne encephalitis virus                                      Russian tick-borne encephalitis                                  Retroviridae type C oncovirus group                                                        type B oncovirus group                                                        type D retrovirus group                                                       avian complex leukemia virus                                                  Rous sarcoma virus                                                            murine complex leukemia virus                                                 mouse sarcoma virus                                                           murine mammary tumor virus                                                    feline leukemia complex virus                                                 feline sarcoma complex virus                                                  woolly monkey sarcoma virus                                                   gibbon leukemia virus                                                         Mason-Pfizer virus                                                            hamster leukemia virus                                                        rat leukemia virus                                                            bovine lymphoma virus                                                         human T cell leukemia viruses:                                                types 1 and 2 etc.                                                            spumaviridae: syncytial and foamy                                             viruses of humans, monkeys, cattle, cats                                      visna virus of sheep                                                          Maedi virus                                                                   progressive pneumonia viruses of sheep                                        *human immunodeficiency viruses:                                              (include HTLV III/LAV) HIV,                                                   HTLV IV,                                                                      LAV-2, STLV-III.sub.AGM                                          Arenaviridae Junin virus                                                                   lassa virus                                                                   machupo virus                                                                 pichinde virus                                                                lymphocytic choriomeningitis virus                                            lassa fever virus                                                             arenavirus                                                       Other virus-like                                                                           kuru virus                                                       agents       Creutzfeldt-Jakob disease virus                                  viroids-prions                                                                             scrapie virus                                                                 transmissible mink encephalopathy                                             Aleutian disease of mink                                                      bovine spongiform encephalopathy "virus"                         ______________________________________                                         *NOTE:                                                                        under Retroviridae human Tlymphotropic virus type III (HTLVIII)               Lymphadenopathy virus (LAV) human immunodeficiency virus (HIV) simian         Tlymphotropic virus type III (STLVIII.sub.AGM)                                human Tlymphotropic virus type IV (HTLVIV)                                    (HTLV III and LAV are now usually referred to as HIV)                    

The term "tumor" as used herein includes both malignant andnon-malignant neoplasms including tumors of viral, chemical, radiation,genetic and other origins. It can be of embryonic ectodermal origin,embryonic mesodermal origin, or embryonic endodermal origin. It can befrom the embryonic surface ectoderm, the embryonic neuroectoderm, theembryonic head mesoderm, the embryonic paraxial mesoderm, the embryonicintermediate mesoderm, the embryonic lateral mesoderm, or the embryonicendoderm. Thus, tumors in an animal include: tumors of the skin and softtissues; tumors of the muscle; tumors and tumor-like lesions of jointsand adjacent soft tissues; tumors of bone and cartilage; tumors of thelymphoid and hematopoietic tissues; tumors of the liver, gall bladderand pancreas; tumors of the urinary system; tumors of the genitalsystems; tumors of the mammary gland; tumors of the endocrine glands;and tumors of the nervous system and eye.

Human malignant tumors include: acute lymphoid leukemia; acute myeloidleukemia; chronic myeloid leukemia; chronic lymphoid leukemia;polycythemia vera; myelosclerosis with myeloid metaplasia; multiplemyeloma; primary macroglobulinemia; Hodgkin's disease; non Hodgkin'slymphoma; skin cancer; malignant melanoma; head and neck cancer; lungcancer; gastointestinal cancer; breast cancer; gynecologic cancer;trophoblastic disease; testicular cancer; prostate cancer; renalcarcinoma; bladder cancer; endocrine tumor; brain tumor; retinoblastoma;neuroblastoma; Wilm's tumor; osteogenic sarcoma; Ewing's sarcoma; andsoft-tissue sarcoma.

The term "microorganism" as used herein includes parasites, bacteria,and other organisms and agents causing infestation. Parasites includearthropod parasites, helminth parasites, protozoal parasites, andhemaprotozoal parasites. Examples of these parasites include demodexmange, hookworm and coccidia.

The term "glycosylation" means the addition of carbohydrate molecules toa protein molecule. An acetylated mannan derivative, in particularacemannan, may exert its therapeutic effect by two possible mechanisms.One is the altering of glycosylation, such as inhibition of glucosidaseI or the incorporation of the acetylated mannan derivative intoglycoprotein. The other possible mechanism is enhancement of theantigenicity of the virus or the tumor, or the enhancement ofimmunocompetency of the host. The enhancement of antigen can be achievedthrough the presentation by macrophage; reception by T or B cells orboth, altered antigen presentation, or adjuvant effect. In a sense,acetylated mannan derivative enhances the recognition of a tumor or ofan infectious agent, such as a virus or another microorganism, as "notself" by the host.

The administration of acetylated mannan derivative can be achieved bytopical application, oral ingestion, IP route, IV route or otherparenteral routes of administration.

Not only can the acetylated mannan derivative be given to the recipientas a single agent, it can also be used in combination with other knowntherapeutic agents that are characterized by their requirement of theparticipation or aid of the host's immune system to achieve theirmaximal therapeutic effect.

Acemannan has now been discovered to be a potent inducer of IL-I andprostaglandin E2 (PGE2) production by human peripheral blood adherentcells in culture. The instant invention is believed to be the firstpractical non-toxic stimulator of IL-1 release. IL-1 is an importantmacrophage product reported in the literature to influence the activityand production of lymphocytes, fibroblasts, B-lymphocytes andendothelial cells. See Old,. Scientific American, 258(5):59-60, 69-75(1988).

IL-1 induces fibroblast proliferation which is fundamental to woundhealing. IL-1 also: (1) enhances bone marrow activity; it may betherapeutic in individuals whose bone-marrow is depressed; and (2)enhances the immune system in general.

A series of experiments with mixed lymphocyte cultures (MLC) has shownthat acemannan increases the alloantigenic response of these lymphocytesin a dose-related fashion. Incubation of acemannan with monocytespermitted monocyte-driven signals to enhance the T lymphocyte responseto lectin. Related studies on acemannan's effects on MLC have shown anincrease in phagocytosis and activity of natural killer cells. Thus, inthese in vitro test systems, acemannan is non-toxic and is animmunoenhancer.

Acemannan actively stimulates lymphocytes to secrete lymphokines andalso causes HIV-infected lymphocytes to produce altered glycoproteins(GP-120) by a mechanism similar to that of glucosidase I inhibitors. SeeGruters et al., Nature 330:74-77 (1987) and Pal et al., Intervirol.30:27-35 (1989). Acemannan is phagocytized and apparently pumped to theGolgi-glycoprotein apparatus of the monocyte where it interferesdirectly with glycoprotein synthesis.

A. Toxicology

The toxicological effects of acemannan have been studies in both in vivoand in vitro systems. Acemannan is not mutagenic or blastogenic in invitro test systems. In vitro, the compound was non-toxic for H-9, MT-2and CEM-SS lymphoid cells. In vivo toxicology studies on acemannaninclude a 91-day subchronic oral toxicity study in dogs, a 180-daychronic oral toxicity study in rats and an 180-day chronic oral toxicitystudy in humans. In these studies, no toxic effects were notes in dogsreceiving up to 825 mg/kg of acemannan per day for 91 days. No clinical,gross pathologic or toxic effects were noted in rats receiving up to38,475 ppm acemannan in their feed for 180 days. No adverse clinical ortoxic effects were notes in human patients receiving 800 mg per day ofacemannan for 180 days.

In pilot studies, administration of acemannan to dogs caused an absolutemonocytosis in blood samples taken for complete white blood cell countsand morphology differential. Within 2 hours after oral administration ofhigh doses of acemannan, large activated monocytes appeared incirculation. A similar effect has been observed in humans.

A study was performed using human peripheral blood monocyte cellcultures and ¹⁴ C-labeled acemannan to track the incorporation orabsorption of acemannan into a biological system. In this study,detectable amounts of ¹⁴ C-labeled acemannan were absorbed or ingestedby human peripheral monocyte/macrophage cells. Peak incorporationoccurred at 48 hours. At a concentration of 5 mg/mg, the ¹⁴ C-labeledacemannan was not cytotoxic to the monocyte/macrophage cells, and theweight/volume (w/v) digested cell mass was 760 times greater than thew/v of the digested acemannan solution. These results suggest that themacrophage is capable of maintaining intracellular concentration ofacemannan at very high levels that are not cytotoxic.

A pyrogen assay was performed in rabbits in accordance with the pyrogentest protocol outlined in the U.S.P. XXI, Biological Test [151], using a1 mg/ml injectable solution of acemannan. More frequent temperaturemeasurements were taken than specified in the U.S.P. because of theunknown systemic effects of injected acemannan. Temperature changes intest animals did not exceed minimum changes allowed by the U.S.P.protocol; therefore, the solution met the U.S.P. requirements forabsence of pyrogens. Acemannan injectable elicited a maximum bodytemperature increase of 0.3° C. in one rabbit. This temperature riseoccurred 90 minutes after injection. Acemannan is an inducer of IL-1secretion by macrophages and monocytes in vitro. Since IL-1 is a potentpyrogen, this might explain the minimal, delayed temperature rise inthis rabbit.

Twenty-four human subjects enrolled in and completed the study of thesafety and tolerance of orally-administered acemannan. Clinicallaboratory results showed that shifts out of the normal range occurredin the following: CO₂ in seven subjects, cholesterol in three subjects,triglycerides in two subjects, phosphorous in one, hemoglobin in four,basophils in two, monocytes in three, eosinophils in three, lymphocytesin four, neutrophils in two, and one each in red and white blood cells.Small numbers of red and white blood cells were also found in the urine.None of these shifts was clinically relevant.

Immune profile results showed group differences between Day 1 to Day 7values for the following: CD-16, CD-4 (T-4), CD-8+Leu7, CD-4+CD-25,CD-8+CD-16, Leu7 and TQ-1. Mitogen responses were in the low range.

Vital signs did not appear to exceed normal ranges. There were no groupdifferences in urine output. One subject in Group IV had diarrhea andloose stools during the study. One subject in Group I had loose stoolsduring days 2to 4 of the study. A total of 5 subjects reported a totalof eight adverse events. All the events occurred in subjects receiving1600 or 3200 mg oral acemannan daily for 6 days.

B. Mode of Administration

The physical properties of acemannan allow it to be formulated andincorporated into all pharmaceutical dosage forms known to those skilledin the art. The biopharmaceutical and toxicological properties ofacemannan permit it to be used in tissues and organs of living organismsand to be administered over a wide range of doses.

Acemannan may be administered to an animal orally, parenterally,topically and locally, in a daily dosage of 0.001 mg/kg to 1000 mg/kgbody weight per day.

Mixed with suitable auxiliaries, acemannan may be compressed or filledinto solid dosage units such as pills, tablets and coated tablets, or itmay be processed into capsules. These oral dose forms would beadministered at a dosage of about 0.1 mg/kg to 1000 mg/kg of body weightper day.

By means of suitable liquid vehicles, acemannan can be injected insolutions, suspensions or emulsions. These products would beadministered at a rate of 0.001 mg/kg to 1000 mg/kg of body weight perday. As an adjunctive component of a vaccine or other product, acemannanwould be used at a rate of 0.001 to 1000 mg per unit dose of adjuvantedproduct.

Topical administration of acemannan can be in the form of a processedgel, cream, lotion, solution, ointment or powder. These formulationscould contain up to 90% acemannan.

EXAMPLE 1 Production of Interleukin-1 and PGE2 By Human AdherentPeripheral Blood Leukocytes Stimulated With Acemannan

A. Induction of IL-1 Production

Human mononuclear cells were separated from heparinized whole blood bydensity-gradient centrifugation in Ficoll-Hypaque (Pharmacia, Sweden).After washing, cells were resuspended at a concentration of 2×10⁶cells/ml in RPMI-1640 with 25 mM Hepes, and supplemented with 50 U/mlpenicillin, 50 μg/ml streptomycin and 2 mM L-glutamine. Two ml aliquotsof the cell suspensions were dispensed into each well of a six-wellplate and incubated for 1 hour at 37° C. in a 5% CO₂ -humidifiedatmosphere. After removal of nonadherent cells, adherent cells werewashed three times with the medium described above. Two ml of mediumsupplemented with 5% pooled human AB serum were added to each well.Cultures were stimulated with acemannan at different concentrations.Simultaneous controls with lipopolysaccharide (LPS) from E. coli (Sigma0111:B4) at a final concentration of 20 μg/ml, and without any addition(background), were included. The cultures were incubated at 37° C. asdescribed above for 24 hours. Supernatants were harvested, centrifugedto remove cells, and dialysed against 500 volumes of PBS for 48 hours(changed once), followed by 4 hours of dialysis against 20 volumes ofRPMI-1640 with 25 mM Hepes, antibiotics and L-glutamine as described.Supernatants were frozen at -20° C. until IL-1 activity was evaluated.

B. IL-1 Determination in Supernatants

Two different procedures were used to assay IL-1: (1) the thymocyteproliferation assay and (2) in ELISA assay specific for IL-1.

1. Thymocytes from C₃ H/HeJ mice 5-8 weeks old were used. A homogeneouscell suspension was prepared in minimum essential medium (MEM)supplemented with 5% FCS, 100 U/ml penicillin, 50 g/ml streptomycin, 2mM L-glutamine and 5×10⁻⁵ M 2-mercaptoethanol. The cell concentrationwas adjusted and dispersed into 96-well plates at 1×10⁶ cells/well.Phytohemagglutinin (PHA) was added to each well at a concentration of 10μg/well. Samples were diluted serially and a volume of 25 μl was addedto each well, starting from 1:10 to the final dilution. Every dilutionwas tested in quadruplicate. Plates were incubated at 37° C. in ahumidified atmosphere with 5% CO₂ for 72 hours and were pulsed with [³H]-thymidine (0.5 μCi/well) during the last 16 hours. Cells wereharvested onto fiberglass filters with an automatic cell harvester, andradioactivity was measured by standard scintillation procedures. Resultsare represented as cpm of thymidine incorporation by thymocytes inresponse to the supernatants at a final 1:10 dilution.

2. Two-site "Sandwich" ELISA for IL-1. This procedure has recently beendescribed in Journal of Immunology, 138:4236 (1987), the disclosure ofwhich is hereby specifically incorporated herein by reference. See alsoU.S. Pat. No. 3,654,090 and U.S. Pat. No. RE 31,006 to Schuurs et al.Briefly, monoclonal purified antibody IL-1-H6 against IL-1β, (100μl/well, 10 μg/ml) was coated on vinyl assay plate wells overnight at 4°C. The wells were washed with PBS/0.5% Thimerosal and countercoated with200 μl of 5% non-fat dry milk/0.5% Thimerosal/PBS for 1 hour at roomtemperature. After washing, 50 μl of sample or human recombinant IL-1standard and 50 μl of another monoclonal antibody against anon-overlapping epitope of IL-1, [biotinylated IL-1β-H67 (2 μg/ml) in 1%non-fat dry milk/0.5% Thimerosal/PBS], were added, and the plates wereincubated for 2 hours at room temperature. After washing, 100 μl /wellof a 1:1000 dilution of streptavidin-peroxidase were added and the platewas incubated for 1 hour. The wells were washed, incubated for 30minutes in the dark with 100 μl OPD substrate solution, and absorbanceat 450 nm was measured.

C. Determination of PGE2

PGE₂ was evaluated with a radioimmunoassay in the same non-dialyzedsupernatants. The antibody to PGE₂ (ICN Biomedical, Inc., Costa Mesa,Calif.) was used according to the manufacturer's instructions, which areincorporated herein by reference.

D. Observations

Representative experiments are shown in Table 2. Acemannan is a potentinducer of IL-1 production by human adherent peripheral bloodleukocytes. At doses between 1 and 10 μg/ml, acemannan extract inducedproduction of IL-1 comparable to that induced by 20 μg/ml LPS, which isthe reference inducer of IL-1 production. Acemannan in the same doserange also induced the production of PGE₂ at levels comparable to thoseinduced by 20 μg/ml LPS (positive control).

                  TABLE 2                                                         ______________________________________                                        INDUCTION OF PGE.sub.2 SYNTHESIS BY HUMAN                                     PERIPHERAL                                                                    BLOOD ADHERENT CELLS STIMULATED BY                                            ACEMANNAN                                                                     AND BY LIPOPOLYSACCHARIDE (LPS).                                              Experiment                                                                    Number     Stimulator      PGE2 (ng/ml)                                       ______________________________________                                        198        0               0                                                             LPS 20 μg/ml 2.6, 3.9                                                      Acemannan 10 μg/ml                                                                         3.5                                                           Acemannan 1 μg/ml                                                                          0                                                  148        0               0                                                             LPS 20 μg/ml 0.5, 1.3                                                      Acemannan 10 μg/ml                                                                         0.7                                                ______________________________________                                    

EXAMPLE 2 Effect of Acemannan on Phagocytoses in Vitro

The effect of acemannan was studied in vitro to ascertain its effect onphagocytic function. CBA mice were injected IP with 1 mg/kg acemannan,and peritoneal and splenic macrophages were collected 3 days later.Thioglycolate and saline were similarly tested as positive and negativecontrols, respectively. The macrophages were incubated with sheep redblood cells (SRBC) as ingestion particles in the presence and absence ofanti-SRBC titers, and phagocytosis was measured histologically aspercent cells that ingested SRBC. Although non-specific phagocytosis wasincreased slightly after acemannan treatment, phagocytosis wassignificantly increased in the presence of antibody. In the presence ofcomplement, acemannan-stimulated, antibody-mediated phagocytosis wasincreased to an even greater extent. These results indicate thatacemannan may increase the number of macrophages and enhance theirphagocytic activity. Such responses may contribute to acemannan'seffectiveness as a stimulant of wound healing and as an anti-infectiousagent.

A. Methods and Materials

Acemannan was stored at room temperature in its dried form. The amountneeded for each experiment was weighed out and microwaved in 2-minuteexposures at 600 watts of power. It was then transferred to a sterileplastic centrifuge tube and microwaved for 1 additional minute. Thematerial was diluted in cell culture medium (RPMI-1640) to the desiredconcentration.

Phagocytic Cells:

Mouse spleen cells were obtained from BALB/c mice purchased from HarlanSprague-Dawley. The mice were killed by CO₂ asphyxiation, and theirspleens were removed aseptically. Subsequently, the cells were separatedinto adherent and non-adherent populations by nylon wool columnfractionation according to the method of Journal of Immunology, 71:220,the disclosure of which is hereby specifically incorporated byreference. Adherent cells were determined by microscopic analysis, asdescribed below, to be macrophages (monocytes) and lymphocytes in aratio of 4 to 1. After single-cell suspensions were obtained by monlayerdisruption, both adherent and non-adherent single cell preparations wereplaced on ficoll-hypaque and centrifuged to obtain a mixture oflymphocytes and macrophages.

Blastogenesis Assay

A standard blastogenesis assay was set up as outlined below. The mitogenused in the assay was PHA-P obtained from Burroughs Wellcome. Asindicated for individual experiments, the cultures were maintained for72 hours in a 5% CO₂, humidified atmosphere. Tritiated thymidine wasadded during the last 6 hours of the culture. Cell concentrations perwell, using flat bottom microtiter tissue culture plates, were 5×10⁵mouse cells/0.2 ml. Cells were deposited in the wells and acemannan ormitogen was added. A stimulation index (SI) was calculated using theformula: ##EQU1##

Cell Staining

Briefly, smears of cells were stained by non-specific esterase stain asfollows. Approximately 2×10⁶ cells in 2 drops were mixed with 2 drops offetal calf serum and 4 drops of a fixative solution consisting of amixture of 25 ml of 35% formaldehyde, 45 ml of acetone, 100 mg of KH₂PO₄, 10 mg of Na₂ HPO₄ and 30 ml of water. The slides were incubatedwith a mixture of 10 mg of naphthyl acetate and 4.5 mg of Fast Bluestain in 1.4 ml of ethylene glycol monomethyl ether with 5 ml of 0.1MTrismaleate buffer, pH 7.8 (Wright's stain). The stain was allowed toreact for 10 minutes, then washed in water for 20 seconds. Acounterstain of 0.2 g of Giemsa stain, 12.5 ml of ethanol and 12.5 ml ofglycerol was used for 30 seconds before washing again.

Induction of Peritoneal Macrophage Cells

Saline thioglycolate broth (1 mg/kg) or acemannan (1 mg/kg) was injectedIP into female BALB/c mice to induce peritoneal exudate macrophagecells. Induced cells were removed from the peritoneal cavity 3 dayspost-injection.

Macrophages were washed twice with phosphate-buffered saline (PBS) andcovered with 2 ml of fresh medium; 0.1 ml of the macrophage suspensionwas added to each tube. Cultures were placed for 30 to 60 minutes into a37° C., humidified 5% CO₂ -95% air incubator. Cultures were washed twicewith PBS and covered with 2 ml of PBS. One of each pair of coverslipswas removed with needle-nosed forceps, dipped for 5 seconds only indistilled water, and promptly replaced in the culture dish. The PBS wasremoved, and the cultures were covered with ice-cold glutaraldehyde.After 10 minutes, the glutaraldehyde was removed, and the coverslipswere overlaid with distilled water.

Mounted coverslips were examined promptly with the oil immersion lens ofa phase contrast microscope. Attachment was scored on the coverslip thatwas not subjected to hypotonic shock, whereas ingestion was scored onthe coverslip that was lysed in distilled water.

Antibody-Dependent and Antibody-Independent Phagocytosis

SRBC, obtained from Austin Biologics Laboratory, Austin, Tex., werewashed three times in PBS (pH 7.2). BALB/c mice were given IP injectionsof 10⁶ cells and bled on day 14 post-injection. Serum was collected,pooled and heat inactivated at 56° C. for 45 minutes. Agglutinationtiters were determined to be 1024 using round-bottomed microtiter wells.

Antibody-independent phagocytosis was determined by incubation of SRBC(0.5% v/v) with macrophages (10⁶) in RPMI-1640 containing 20% fetal calfserum (FCS). Slides were prepared at various intervals and stained. Thepercent macrophages that had ingested red cells was determined visuallyby counting 200 cells/slide and three slides/animal.

Antibody-dependent phagocytosis was determined using SRBC (0.5% inRPMI-1640 with 20% FCS) mixed with anti-SRBC serum or IgM fraction(minimum titer of 2000). The mixture was incubated for 15 minutes at 37°C., then washed twice in PBS (pH 7.20 and resuspended to the originalvolume.

Serum Fractionation

Whole serum was fractionated to remove IgM by euglobulin precipitationand dialysis against distilled water. After dialysis at 4° C. for 24hours, precipitate was removed by centrifugation at 1500×G for 20minutes, and supernatant was analyzed by ion electrophoresis andcomplement-mediated lysis. Less than 5% of the original IgM remained.

B. Results

To evaluate the effect of acemannan on macrophages, the first experimentutilized mouse spleen cells cultured in vitro with acemannan (Table 3).

                  TABLE 3                                                         ______________________________________                                        PERCENT CELL TYPES BY HISTOLOGICAL                                            EVALUATION OF MOUSE SPLEEN CELLS IN CULTURE                                   Time in            Acemannan (μg/well)                                     Culture                                                                              Cells (a)   0.0      0.002 0.02   0.2                                  ______________________________________                                        72 hours                                                                             macrophages 30 ± 6                                                                              32 ± 7                                                                           41 ± 3                                                                            45 ± 9                                   lymphocytes 70 ± 5                                                                              68 ± 8                                                                           59 ± 3                                                                            55 ± 6                            96 hours                                                                             macrophages 22 ± 4                                                                              28 ± 4                                                                           36 ± 6                                                                            38 ± 8                                   lymphocytes 78 ± 8                                                                              72 ± 7                                                                            64 ± 10                                                                          62 ± 4                            ______________________________________                                         (a) Macrophages (monocytes) were determined by esterase staining. The         results are expressed as mean ±S.D. The results are from six               experiments with 200 cells studied/experiment. "Lymphocytes" are cells        that did not stain by esterase and had the appearance of lymphocytes by       Wright's stain.                                                          

Cultures were incubated for 72 or 96 hours, and at termination of theexperiment smears were made and stained by Wright's stain and by theesterase method. The relative percentage of macrophages and lymphocyteswas determined. At 72 hours there was a dose-related increase inmacrophage numbers from 30% with no acemannan to 45% with 0.2 μg ofacemannan per well. Since data are expressed as percent cells, there wasa concomitant reduction in lymphocytes. At 96 hours there was also adose-related increase in the percentage of macrophages in the presenceof acemannan. At 96 hours, the cultures with 0.2 μg of acemannan perwell showed significant acidosis, as indicated by a yellow coloring.Furthermore, 96-hour cultures had a lower percentage of macrophages,possibly due to the longer time in culture. To relate theacemannan-induced increase in macrophage numbers to a known standard, asimilar experiment was conducted with the mitogen PHA-P. Results areshown in Table 4.

                  TABLE 4                                                         ______________________________________                                        PERCENT CELL TYPES BY HISTOLOGICAL                                            EVALUATION OF MOUSE SPLEEN CELLS IN CULTURE                                   Time in            PHA-P (μg/well)                                         Culture                                                                              Cells (a)   0.0      0.02  0.01   0.2                                  ______________________________________                                        72 hours                                                                             macrophages 33 ± 8                                                                              32 ± 6                                                                           30 ± 6                                                                            31 ± 5                                   lymphocytes 70 ± 12                                                                             68 ± 8                                                                           70 ± 6                                                                            69 ± 4                            96 hours                                                                             macrophages 18 ± 6                                                                              21 ± 3                                                                           26 ± 6                                                                            25 ± 5                                   lymphocytes 77 ± 10                                                                             79 ± 4                                                                           74 ± 8                                                                            75 ± 6                            ______________________________________                                         (a) Monocytes were determined by esterase staining. The results are           expressed as mean ±S.D. The results are from six experiments.              "Lymphocytes" are cells that did not stain by esterase and had the            appearance of lymphocytes by Wright's stain.                             

Although the percentage of macrophages did not change at 72 hours, therewas a dose-related increase in macrophages after incubation with PHA-Pfor 96 hours. By comparison, acemannan was twice as effective as PHA-P.The percentage of macrophages increased a maximum of 16 with acemannancompared to 7 with PHA-P (Tables 3 and 4).

Since acemannan appeared to increase the percentage of macrophages, itwas decided to determine whether the activity of the phagocytes was alsoincreased. Peritoneal exudate cells from CBA mice given saline,thioglycolate broth or acemannan were used [as phagocytes] with sheepred blood cells as the particles to b ingested (Table 5).

                                      TABLE 5                                     __________________________________________________________________________    NONSPECIFIC PHAGOCYTOSIS OF SHEEP                                             ERYTHROCYTES BY PERITONEAL EXUDATE (a)                                        Percent of Phagocytosis (b)                                                   Time (minutes)                                                                Trmt.                                                                              0   5    10    20    60     120                                          __________________________________________________________________________    Saline                                                                             3 ± 3                                                                          11 ± 6                                                                          15 ± 10                                                                          25 ± 9                                                                           45 ± 12                                                                           52 ± 15                                   Thio.                                                                              1 ± 1                                                                          14 ± 8                                                                          20 ± 8                                                                           52 ± 14 (c)                                                                      84 ± 32 (c)                                                                       99 ± 21 (c)                               Ace. 3 ± 2                                                                          10 ± 6                                                                          12 ± 8                                                                           41 ± 18                                                                          61 ± 18                                                                           63 ± 23                                   __________________________________________________________________________     (a) The results were determined by counting 200 cells/slide with two          slides/animal. The results are based on two experiments.                      (b) Percent phagocytosis indicates the proportion of cells showing            erythrocyte ingestion. The results are expressed as mean ±S.D.             (c) Significantly different from saline control group, assessed by the        Student's ttest at the 95% confidence level.                             

Over a 120-minute period, nonspecific phagocytosis increased from 3% to52% in saline controls, whereas percent phagocytosis in cells fromthioglycolate broth-treated animals rose to 89%. Phagocytosis inacemannan-treated animals rose to 63% at 120 minutes.Acemannan-stimulated phagocytosis was greater than that in controlsafter 20-120 minutes; however, the differences were not statisticallysignificant.

To determine whether the acemannan effect on phagocytosis wasantibody-dependent, a similar experiment was performed with anti-SRBC(Table 6).

                                      TABLE 6                                     __________________________________________________________________________    ANTIBODY MEDIATED PHAGOCYTOSIS (a)                                            Phagocyte      Antibody Titer (× 103) (b)                               Source Pretreatment                                                                          0     2      4     8                                           __________________________________________________________________________    Peritoneum                                                                           Saline  15 ± 8                                                                           43 ± 10                                                                           39 ± 9                                                                           19 ± 11                                         Thioglycolate                                                                         49 ± 11                                                                          89 ± 22                                                                           80 ± 22                                                                          58 ± 14                                         Acemannan                                                                             36 ± 14                                                                          73 ± 13 (c)                                                                       62 ± 8                                                                           40 ± 13                                  Spleen Saline  11 ± 4                                                                           38 ± 9                                                                            32 ± 11                                                                          20 ± 4                                          Thioglycolate                                                                         29 ± 9                                                                           73 ± 3                                                                            54 ± 16                                                                          38 ± 12                                         Acemannan                                                                             21 ± 10                                                                          60 ± 9 (c)                                                                        51 ± 17                                                                          26 ± 11                                  __________________________________________________________________________     (a) Phagocytosis is expressed as the mean % of cells showing erythrocyte      ingestion ±S.D.                                                            (b) The antibody titer by agglutination was shown to be 1:1024.               Pretreatment and cell sources are discussed in Methods.                        (c) Significantly different from saline control, assessed by the             Student's ttest at the 95% confidence level.                             

Sera were inactivated with heat (56° C. for 30 minutes), and theantibody titer used was 2×10³, well above the hemagglutination titer. Inthis experiment, macrophages were obtained from two sources, theperitoneal cavity and the spleen. Again, mice were pretreated with IPinjections of 1 ml of 0.9% saline, 1 mg/kg thioglycolate or 1 mg/kgacemannan. At a titer of 2×10³, the phagocytic activity ofthioglycolate-induced peritoneal macrophages was twice as great (89% vs.43%) as activity from the saline-induced controls, whereasacemannan-induced macrophages were more active by 30% (73% vs. 43%)compared to controls. The difference between phagocytic activity in theacemannan-treated and saline control groups was statisticallysignificant.

Similar results were seen with macrophages obtained from mouse spleens.Phagocytic activity was lower than that of macrophages obtained from theperitoneal cavity, possibly due to manipulations of the spleen cells.Again, at a titer of 2×10³, acemannan-induced macrophages weresignificantly higher in phagocytic activity than saline controls at the95% confidence level; phagocytic activity was similar to control at atiter of 8×10³.

To determine the effect of complement (C') on antibody-mediatedphagocytosis, an experiment utilizing addition of C' to media wasundertaken. (Table 7).

                  TABLE 7                                                         ______________________________________                                        COMPARISON OF COMPLEMENT-MEDIATED                                             PHAGOCYTOSIS                                                                  % Phagocytosis (a)                                                            Cell Source Phagocyte   Inducer +C'                                                                              -C'                                        ______________________________________                                        Peritoneum  Saline      24 ± 11 18 ± 9                                              Thioglycolate                                                                             84 ± 10 62 ± 12                                             Acemannan   70 ± 8 (b)                                                                            54 ± 4                                  Spleen      Saline      18 ± 11 16 ± 9                                              Thioglycolate                                                                             54 ± 9  41 ± 11                                             Acemannan   48 ± 10 35 ± 6                                  ______________________________________                                         (a) Phagocytosis is measured as percent uptake of sheep erythrocytes          ±S.D. after incubation for 30 minutes. Guinea pig complement was added     (b) Significantly different compared to -C', assessed by the Student's        ttest at the 95% confidence level.                                       

To assure that lysis would not occur, IgM-depleted mouse serum was used(see Methods). The titer utilized was 3000, as determined byhemagglutination and the Coombs technique. Cells from both theperitoneal cavity and spleen were more active in phagocytosis with theaddition of C' than without C', although the difference wasstatistically significant only with peritoneal cells induced byacemannan.

Finally, an experiment was performed to differentiate the effect ofacemannan phagocytosis and adherence (Table 8).

                  TABLE 8                                                         ______________________________________                                        COMPARISON OF PHAGOCYTOSIS AND                                                ADHERENCE (a)                                                                 Cell                                                                          Source (b)                                                                             Pre-treatment                                                                             Phagocytosis                                                                              Adherence                                    ______________________________________                                        Peritoneum                                                                             Saline       5 ± 9    6 ± 4                                             Thioglycolate                                                                             12 ± 9   23 ± 9 (c)                                         Acemannan   11 ± 9   18 ± 10 (c)                               Spleen   Saline       8 ± 7   14 ± 11                                            Thioglycolate                                                                             14 ± 6   36 ± 10 (c)                                        Acemannan   10 ± 8   20 ± 7 (c)                                ______________________________________                                         (a) Cell mixtures were allowed to incubate for 7 minutes.                     (b) Results are reported as percent phagocytes showing phagocytosis or        adherence ±S.D. The results are from one experiment with 200 cells         scored/animal with three animals used.                                        (c) Significantly different from saline controls, assessed by the             Student's ttest at the 95% confidence level.                             

In this experiment, antibody to SRBC was used in a titer of 2,000, butthe experiment was stopped after 7 minutes. Acemannan-inducedmacrophages from both the peritoneum and spleen were more efficient inadherence that the saline controls and, as seen previously, lessefficient than the thioglycolate-induced group.

C. Discussion

The results indicate that acemannan both directly and indirectlystimulates phagocytosis. The results also indicate that acemannanenhances phagocytosis by macrophages, both non-specifically andspecifically, through antibody-mediated reactions. This demonstratesthat acemannan has immunostimulatory properties on phagocytes.

EXAMPLE 3

The Effects of Acemannan on Nonspecific Tumor Lysis

This example investigates the possibility of nonspecific tumor deathinduced by acemannan-stimulated phagocytes.

A. Procedures

Acemannan Polymer

Acemannan was kept in a dried form. The amount needed for eachexperiment was weighed and microwaved in 2-minute exposures at 600 wattsof power. The material was transferred to a sterile centrifuge tube (15ml) and microwaved for one additional minute. The material was dilutedin Hanks Balanced Salt Solution (HBSS) to the concentration needed. Insome experiments, material was sterilized by autoclaving, with noapparent loss in activity.

Cells

Macrophages were harvested from the peritoneal cavity of BALB/c femalemice obtained from Harlan/Sprague Dawley. Either thioglycolate broth (25mg/kg) or acemannan (25 mg/kg) was injected IP into some groups ofanimals 6 days before harvesting. Saline stimulated cells were alsoutilized as an additional control. Harvested cells were washed threetimes in HBSS and diluted in RPMI-1640 to a concentration of 5×10⁶/cells/ml.

Target cells

Target cells were obtained from the American Type Culture Collection(C3H/HeN Fibrosarcoma L929) and maintained in passage. Labeling was donewith 150 mCi of ⁵¹ Cr mixed with 1 ml of the cell suspension containing10⁷ cells in RPMI-1640. Cells were incubated for 1 hour, washed withRPMI-1640 three times and adjusted to a final concentration of 5×10⁴cells/ml.

B. Assay

Aliquots of effector cells (100 cells/μl) were placed in flat-bottomedmicrotiter plates. ⁵¹ Cr-labeled cells were added with a minimum ofthree replicates per experimental point. Test plates were incubated at37° C. in 7% CO₂ (previously 5% CO₂) for 20 hours. Supernatants (100μl), were obtained after centrifugation of the plates at 250×G for 15minutes. The amount of radioactivity was assayed on a Packard gammacounter. Controls consisted of thymocytes. The percent of cytotoxicity(% CT) was determined by: ##EQU2##

C. Results

Table 9 shows the results of the initial experiments.

                  TABLE 9                                                         ______________________________________                                        EFFECT OF ACEMANNAN ON CYTOTOXICITY                                                                        percent                                          Cells            cpm ± S.D. (a)                                                                         cytotoxicity                                     ______________________________________                                        thioglycolate stimulated                                                                       2,800 ± 300                                                                            6.6                                              in vivo                                                                       thioglycolate stimulated                                                                       2,950 ± 260                                                                            7.0                                              in vitro                                                                      nonstimulated    2,870 ± 400                                                                            6.8                                              Acemannan-stimulated                                                                           3,100 ± 360                                                                            7.4                                              in vivo                                                                       Acemannan-stimulated                                                                           21,000 ± 900                                                                           50.0                                             in vitro                                                                      Acemannan-stimulated                                                                           20,500 ± 1100                                                                          48.8                                             in vivo and in vitro                                                          ______________________________________                                         (a) total cpm of target cell = 42,000                                    

Thioglycolate-stimulated macrophages incubated with ⁵¹ Cr target cellsrelease ⁵¹ Cr at an average of 2800 cpm, whereas acemannan-labeled cellsreleased radioactivity at an average of 3100 cpm. There was nostatistical difference between these groups. Nonstimulated macrophagesreleased in the range of 2800 cpm. However, macrophages stimulated withacemannan in vitro had a ⁵¹ Cr release of 21,000 cpm. This indicates twothings: 1) acemannan does not induce a long standing cytolytic effect,and 2) its activation can occur in a relatively short time in tissueculture. The percent cytotoxicity is parallel to the cpm released fromtarget cells when destroyed.

A subsequent experiment using the cytotoxic assay over time is shown inTable 10.

                  TABLE 10                                                        ______________________________________                                        TIME DEPENDENT EFFECT OF ACEMANNAN ON                                         CYTOTOXICITY                                                                  Percent                                                                       Time (a) Stimulation  cpm (b)  Cytotoxicity                                   ______________________________________                                        0        Acemannan      800    2.0                                                     Thioglycolate                                                                                780    1.9                                            3        Acemannan     1,400   3.5                                                     Thioglycolate                                                                                800    2.0                                            6        Acemannan    18,000   46.0                                                    Thioglycolate                                                                               1,200   3.0                                            9        Acemannan    22,600   57.9                                                    Thioglycolate                                                                               2,200   5.8                                            12       Acemannan    22,500   57.6                                                    Thioglycolate                                                                               2,300   5.8                                            15       Acemannan    23,000   58.9                                                    Thioglycolate                                                                              21,100   5.8                                            ______________________________________                                         (a) Time in hours after injection                                             (b) Cpm control cells = 39,000                                           

The cytotoxic effect of acemannan began within 6 hours after stimulationand increased to its maximum by 9 hours. The mechanism of thisactivation has not been investigated.

The data shown in this example indicate that acemannan may have animportant role in the nonspecific therapy of cancer.

Screening of Acemannan for Potential Efficacy Against Equine Sarcoid.

Three sarcoids on two horses were treated both parenterally andintralesionally with acemannan. The goals of this trial were todetermine whether acemannan might be an effective treatment againstequine sarcoid and also to observe the horses for adverse reactions. Onhorse 1, one sarcoid completely resolved while a second sarcoid did notdecrease in size. A third nodular sarcoid developed during treatment. Onhorse 2, a single sarcoid completely resolved. These results suggestthat acemannan may be useful in the treatment of equine sarcoid.

Two horses with three suspicious lesions were purchased at a sale. Thelesions were photographed, measured and confirmed by histopathology assarcoids.

Horse 1

Day 1. Each of the two lesions on the right rear leg was treated bydirect injection 20-ga. needle), with 50 mg acemannan diluted in 10 mlsaline (lesion 1) and 5 ml saline (lesion 2). Twenty-five mg acemannandiluted in 7.5 ml saline was also given IV.

Day 7. Lesion 1 (upper lesion) was treated (18 ga. needle) with 50 mgacemannan diluted in 10 ml saline. Lesion 2 was treated with 25 mgdiluted in 7.5 ml saline. Fifty mg in 10 ml saline was given IV.

Day 14. Lesion 1 was treated with 50 mg in 10 ml saline, whereas lesion2 was treated with 25 mg in 5 ml saline. Seventy-five mg in 25 ml salinewas given IV.

Day 21. Lesion 1 was treated with 50 mg in 10 ml saline, and lesion 2was treated with 25 mg in 10 ml saline. One hundred mg in 25 ml salinewas injected IV.

Day 29. Lesion 1 was treated as on day 21, but because of local swellinglesion 2 was not treated directly. One hundred mg in 25 ml saline wasgiven IV.

Day 42. Lesion 1 was not treated directly. Lesion 2 was treated with 25mg in 10 ml saline. One hundred mg in 50 ml saline was given IV.

Day 57. Horse 1 was euthanized. Tissue samples were taken at the site oflesion 1 and from lesion 2, inguinal lymph nodes and a nodular lesion onhis left shoulder that had developed during the course of treatment.

Horse 2

Day 1. The lesion on the lower left thorax was treated with 50 mgacemannan diluted in 30 ml saline. One half was injected subcutaneously(S/Q) and the other half intralesionally.

On days 6, 16 24, 30, 49, 56, 63, 70 and 77 horse 2 was given 100 mgacemannan IV diluted in 60-120 ml saline, the amount of diluent varyingas required to make a clear solution.

On days 105, 113 and 120, the lesion was treated with 25 mg acemannandiluted in 5 mg saline, intralesionally and S/Q at the base of thelesion. An additional 75 mg was given IV.

Results-Horse 1

Day 1. Lesion 1 measured 2.5 cm (length horizontally)×2.5 cm (heightvertically)×1 cm (thickness). The resolution of this lesion can befollowed below:

    ______________________________________                                        Horse 1 - Lesion 1                                                            Day       Measurements                                                        ______________________________________                                         1        2.5 cm × 2.5 cm × 1 cm                                   7        2.5 cm × 1.75 cm × 1 cm                                 14        2.0 cm × 1 cm × 1 cm                                    21        2.0 cm × 1 cm × now flush with skin level               29        2.0 cm × 1 cm × flat and dry                            42        all but healed                                                      54        completely healed                                                   ______________________________________                                    

Lesion 3 measured 2 cm×2 cm×1 cm on Day 1 and never changedsignificantly.

    ______________________________________                                        Horse 1 - Lesion 2                                                            Day     Measurements                                                          ______________________________________                                         1      2 cm × 2 cm × 1 cm                                         7      2 cm × 2 cm × 1 cm                                        14      2 cm × 2 cm × 1 cm                                        21      2 cm × 2 cm × 1 cm                                        29      2 cm × 2 cm × 1 cm - entire hock still swollen                    and painful                                                           42      size slightly less - still swollen, not as                                    painful                                                               54      same size - hock swelling down 65%                                    ______________________________________                                    

Results-Horse 2

Day 1. The lesion measured 5 cm×3.5 cm×2.5 cm with a pedunculated baseof 2.5 cm. The changes until complete resolution are shown below:

    ______________________________________                                        Horse 2 - Lesion 1                                                            Day          Measurements                                                     ______________________________________                                         1           5 cm × 3.5 cm × 2.5 cm                                6           no change                                                        16           no change - more granulomatous                                   24           5 cm × 3 cm × 2.5 cm                                 30           less granulomatous                                               49           4 cm × 3 cm × 2 cm                                   56           4 cm × 3 cm × 2 cm                                   63           3.8 cm × 3 cm × 2 cm                                 70           3.7 cm × 2.6 cm × 1.8 cm                             77           2.7 cm × 2 cm × 1.3 cm                               105          2.5 cm × 2 cm × 1.3 cm                               113          3.5 cm × 2.25 cm × 1.5 cm                            120          2.5 cm × 2.4 cm × 0.6 cm                             177          Lesion completely resolved                                       ______________________________________                                    

After IV administration to these horses, there were no changes in heartrate, no sweating, muscle fasciculation or obvious signs of distress. Aslight increase in depth of respiration was noted in horse 1 only.Locally, horse 1 showed an inflammatory cellulitis of a mild nature atlesion 1 and of an acute painful type at lesion 2, enough so that thelesion was not injected as scheduled on day 29. Lesion 2 was morefibrous and much more difficult to inject, so that there was moreleakage S/Q. This could account for the lack of effect on lesion 2.Horse 2 did not show cellulitis.

The fact that a nodular sarcoid developed during the course of treatmentleads one to suspect that the main effect of acemannan is a local tissuereaction rather than a systemic one, although IV administration maysensitize the sarcoid to intralesional treatment.

The exact data at which the lesion on horse 2 resolved is unknownbecause the investigator was on a 60-day sick leave between day 113 andday 177. Judging from the lack of significant reduction in tumor size byday 56, it would appear that weekly IV administration alone had littleeffect on the sarcoid on horse 2.

EXAMPLE 4 Enhancement of Allo-Responsiveness of Human Lymphocytes byAcemannan

This example was designed to test the capacity of acemannan to enhanceimmune response to alloantigen and to test whether the potentialenhancement is a monocyte-driven phenomenon. Acemannan did not enhancelymphocyte response to syngeneic antigens in the mixed lymphocyteculture (MLC), but, importantly, it increased alloantigenic response ina dose-response fashion (2.6×10⁻⁷ --2.6×10⁻⁹ M). This effect ofacemannan was shown to be a specific response and to concur withconcentrations of in vitro acemannan achievable in vivo. A separateseries of mixing experiments demonstrated that acemannan incubation withmonocytes permitted monocyte-driven signals to enhance T cell responseto lectin. It is concluded that acemannan is the active ingredient ofthe Aloe vera plant and is an important immunoenhancer in that itincreased lymphocyte response to alloantigen. It is suggested that themechanism involves enhancement of monocyte release of IL-1 under theaegis of alloantigen. This mechanism may explain in part the capacity ofacemannan to abrogate viral infections in experimental animals and man.

This example was designed to directly assess the impact of acemannan asan immune enhancer in the model of monocyte-T-lymphocyte, cell-cellinteraction response to alloantigen presented in the mixed lymphocyteculture. This model tests the capacity of acemannan to stimulateadditional monocyte-macrophage functions in an immunologically relevantmodel.

A. Materials and Methods

1. Cell Preparation

Mononuclear leukocytes were obtained from the peripheral blood ofnormal, informed and consenting human volunteers under the aegis of astudy approved by the Institutional Review Board of the University ofTexas Southwestern Medical Center at Dallas. Peripheral blood wasdiluted 1:3 in Hanks' balanced salt solution (HBSS) and layered on topof a ficoll-hypaque gradient. Cells from subjects known to be majorhistocompatibility disparate were obtained on each study day to ensure apositive mixed lymphocyte reaction. For specific experiments, morecarefully characterized pedigrees of cells which inhibit the mononuclearleukocyte pool were isolated. T-lymphocytes were isolated by thestandard nylon wool separation technique. The nylon effluent cellscontained about 90% pure T cells. T-8 lymphocytes andmonocyte-macrophages preferentially adhere to the column. The adherentpopulation was removed by forcibly pushing media through the column witha plunger. To enrich for monocytes (macrophages), the glass adherenceprocedure was utilized to produce a population greater than 95% pure.

2. Acemannan.

Acemannan was tested in these studies by preparing a 0.5% (w/v) solutionin RPMI-1640 medium and further diluting to the following workingconcentrations:

    2.6×10.sup.-7 M, 2.6×10.sup.-8 M and 2.6×10.sup.-9 M

3. Mixed Lymphocyte Cultures (MLC).

Unidirectional MLC were set up in microtiter, flat-bottom tissue cultureplates (Costar Co., Cambridge, Mass.). Mononuclear cells, isolated bythe ficoll-hypaque density gradient technique discussed above, served asstimulator cells after exposure to 2000 rads for 30 minutes in a cesiumsource (Gammacell, Atomic Energy of Canada, Ontario, Canada). Respondercells that had been similarly isolated and stimulators were adjusted to1.3×10⁶ cells/ml. To each well the following were added: 25 μl ofacemannan or media (control), 25 μl of RPMI-1640 supplemented with 10%fetal bovine serum and 75 μl of each cell population. Cells wereincubated at 37° C. in 5% Co₂ : 95% air for 6 days. Cultures were pulsedwith 25 μl of ³ H-thymidine (1 μCi/well) for 4 hours, after which thecells were harvested and counted. To test the specificity of acemannanon the afferent recognition and response to MLC, additionalunidirectional MLC were set up with the agent added just 20 minutesbefore the cells were pulsed with ³ H-thymidine.

4. Monocyte-T Cell Interaction

Lewis female rat spleens were teased through a sterile steel mesh intoRPMI-1640 medium. Mononuclear leukocytes were collected from theinterface of a ficoll-hypaque density gradient as described above.Monocytes,obtained by enrichment on glass petri dishes and adjusted to afinal concentration of 10⁶ /ml, were incubated with varying doses ofacemannan or medium (control) in a total volume of 2 ml and incubatedfor 24 hours at 37° C. The monocytes were harvested, extensively washedwith fresh medium and co-cultured with syngeneic T lymphocytes at aratio of 10 T-cells; 1 monocyte, with the plant lectinphytohemagglutinin (Difco, Detroit, Mich.) (1:100) for 48 hours at 37°C. Cells were harvested over a MASH II (Whittaker, Mass. Bioproducts,Walkersville, Md.), placed in fluor and counted in a scintillationcounter (Beckman Laboratories, Chicago, Ill.). A control experiment wasperformed by incubating T lymphocytes with acemannan, followed by washand co-culture with freshly acemannan, followed by wash and co-culturewith freshly prepared T lymphocytes, again at 10:1 along with PHA-P.

B. Results

1. Alloantigenic Response

Acemannan had no statistically important effects on the response ofT-cells to autoantigens. When the agent was added at the beginning ofMLC, cells receiving syngeneic stimulation incorporated tritiatedthymidine equally in the presence or absence of test reagent at thedoses described. In the absence of oral acemannan these MLC incorporated2616±1099 cpm of tritiated thymidine at the end of a 4 hour pulse.Although there was a trend upward with respect to the dose of agentadded (3281±1355 at 2.6×10⁻⁹ M, 3742±1670 at 2.6×10⁻⁸ M, and 3828±1978at 2.6'10⁻⁷ M), none of these rates of isotopic incorporation into DNAwas different to a statistically significant degree.

In contrast to the absence of effect of acemannan on autoreponse in theMLC was the agent's effect on alloresponse in the same immunologicassay. First, acemannan did not interfere with the capacity oflymphocytes to recognize and respond to class II alloantigenicdifferences in the MLC; this was apparent when the syngeneic cultureswere compared to the allogeneic response in the presence of the lowestconcentration of drug. Second, there was a dose-response-relatedenhancement of alloresponse by acemannan such that the culture treatedwith the highest dose, 2.6×10⁻⁷ M, reflects a nearly 60% increase overthe non-acemannan culture. The dose response relationship is mostconvincingly demonstrated as the enhanced allogeneic response is shownto be significant for each dose of acemannan tested with respect to theno acemannan condition.

To ascertain whether acemannan exerts a specific effect on lymphocytealloresponse or a nonspecific effect on tritiated thymidineincorporation, the reagent was added at the conclusion of a 7 day mixedlymphocyte culture MLC, 20 minutes before addition of the tracer to theculture. There was no effect of acemannan when added in this manner as apulse at the conclusion of the MLC. These data support the specificityof the acemannan effect on enhancement of lymphoid response in the MLC.

2. Acemannan and Monocyte-T Cell Cooperation.

To test hypothesis that acemannan directly stimulates the monocyteresponding to alloantigen to provide signal(s) to enhance lymphoidresponse to antigen and/or mitogen, purified populations of monocyteswere incubated for 24 hours with various doses of acemannan. At theconclusion of the incubation the cells were washed extensively and thenco-cultured with T lymphocytes at a ratio of 10:1, to stimulate thenatural ratio found in peripheral blood. Co-cultured cells werestimulated with phytohemagglutinin. The co-cultures with monocytes thatwere previously incubated with acemannan had a significantly increasedmitogenic response in a dose-related fashion.

C. Discussion

This example has explored the capacity of acemannan to function as animmunostimulating drug with important clinical consequence.

Acemannan is believed to be capable of limiting DNA and retrovirusinfections that cause significant diseases in animals and in man. Forexample, in an animal model, acemannan ameliorated feline viralrhinotrachetis. Additional evidence shows that acemannan in vitro and invivo may be effective against Herpes simplex II virus, the measlesvirus, and perhaps Herpes simplex II virus, the measles virus, andperhaps HIV. Evidence indicates that the immunological mechanism mayinvolve enhancement of the monocyte, both as a phagocytic cell and as acell that contributes to afferent recognition of antigen. Studies haveshown direct enhancement of phagocytic properties of the monocyte, onthe one hand, and an increase in the absolute numbers of that importantcell, on the other. Mounting evidence supports the concept thatacemannan enhances the elaboration by the activated monocyte of thesignal substance IL-1.

The studies described in this example were directed specifically atexploring the mechanism by which acemannan may be an immuno-enhancingreagent. Mixed lymphocyte cultures are in vitro models of the manner inwhich immunocompetent cells participate in response to antigen of thevariety that is necessary for recognition and response to virus. In thisreaction, there are important monocyte-T-lymphocyte interactions thatgenerate a response to alloantigen. It wash this model that was chosenfor testing the capacity of the acemannan to function as animmunoactivator.

Acemannan is therefore an important enhancer of the alloantigenicresponse in MLC. There is a dose-response relationship with enhancementat the highest dose tested of about 60% above basal. This represents notonly a statistically significant but also a biologically relevantincrease in response to alloantigen and may serve as one means by whichthe drug can aid the response of the organism to viral assault. Thiseffect of acemannan was shown to be specific for the allogeneicstimulus, provided the drug did not enhance either basal response toself (syngeneic MLC) or non-specific incorporation of a tracer DNAprecursor, tritiated thymidine, when drug was added at the conclusion ofthe MLC.

A second series of experiments tested the hypothesis thatmonocyte-T-lymphocyte interactions may be, at least in part, responsiblefor the heightened alloresponse in the MLC. In this series ofexperiments acemannan was incubated along with monocytes, after whichthe treated, extensively washed monocytes were mixed with freshlyprepared, syngeneic T-lymphocytes that had not been exposed to and wouldnot be exposed to acemannan. These experiments demonstrate theenhancement of T-lymphocyte response to the polyclonal mitogenphytohemagglutinin at a magnitude equal to the response that had beenseen previously in the MLC--approximately 55% above baseline anddose-response relationship.

The lowest dose that was tested in the study that was effective in theMLC had no effect i the monocyte experiment. It is not surprising thatthe threshold dose may be different for the two models tested,polyclonal response to mitogen and alloantigenic response in the MLC. Itcan also be observed that the monocyte experiment is a more stringenttest of the effect of acemannan because it presents a treated cell type,the monocyte, to T cells that then see an immune stimulus in the absenceof the drug. While the alloantigenic response may be due solely or ingreat measure to acemannan-enhanced monocyte production of IL-1, thelesser polyclonal mitogen-enhanced response may be a consequence of anassay of immune stimulations, each with a different threshold responseto acemannan.

The dose of acemannan used in these experiments is clinically relevant.The dose range selected was chosen precisely to bracket thatconcentration of acemannan that could be expected to be achieved inplasma if the drug distributes in extracellular water and is absorbed atthe rate of a third of the orally administered dose, figures that werebased on previous pharmacologic studies in dogs. The actualconcentrations achievable in man have also bee shown to be in thisrange, further supporting the potential relevance of these studies forclinical practice.

Acemannan was shown by these experiments to cause monocytes to releasemonocyte-driven signals to enhance T4 cell response to lectin. Whileacemannan did not enhance lymphocyte response to syngeneic antigens inMLC, it did increase MLC alloantigenic response in a dose-relatedmanner. This response was shown to be an acemannan-specific response atacemannan concentrations achievable in vivo.

This experimental documentation demonstrates that acemannan is animmunoenhancer and biological response modifier in that it increaseslymphocyte response to alloantigen. A proposed mechanism of actioninvolves stimulation of monocytes to release IL-1; in the presence ofacemannan, IL-1 has been shown to be released from monocyte cultures.The pharmacologic action of acemannan stimulation of monocytes mayexplain acemannan activity against viral infection in animals and man.

EXAMPLE 5 Pharmacokinetic Basis For Correlation of In Vitro and In VivoEffectiveness of Acemannan

To evaluate the pharmacokinetic behaviour of acemannan, ¹⁴ C-labelledmaterial was given by IP and IV injection and PO administration. Basedon the results of previous pilot work, an aqueous dose of 200 mg ¹⁴C-labelled acemannan/200 ml with specific activity of 17.4 cpm/μg wasadministered to female dogs (approximately 20 mg/kg). Blood, urine andfeces samples were taken at appropriate intervals for 48 hours orlonger. Organ and tissue samples were taken after sacrifice, and allsamples were analyzed for radioactivity using scintillationspectrometry.

Acemannan's kinetic behaviour was typical of that seen with mostpharmacologic agents; however, its biologic half-life (t_(1/2)) wasextraordinarily long. Significant absorption occurred by all threeroutes of administration. Maximum blood levels were achieved after IVinjection followed by IP and then PO. Blood levels, which wereimmediately maximal at 200 μg/ml after IV injection, declined with at_(1/2) of 50-60 hours; plasma levels were approximately twice those ofblood. By comparison, after IP injection blood levels peaked at 45 μg/mlat 24 hours and then declined at a rate similar to that seen with IV; infact, blood levels were nearly 90% maximal after only 8 hours. With oraladministration, blood levels were measurable after 3 hours and peaked at4-5 μg/ml. Based on the relatively long half-life in blood, atherapeutic dosing interval of approximately 7 days would be justified,considering the time required for three half-lives.

Radiolabeled acemannan distributed mainly in liver and spleen followingIP or IV injection. Liver, marrow, thymus, and lymph nodes were primarysites of distribution after oral dosing, a finding consistent with theimmunologic sites of action for acemannan. Levels of radiolabeledcompound in tissues sampled after 48-52 hours ranged from a low ofapproximately 1 μg/g brain to a high of 85 μg/g spleen after IVinjection. Interestingly, levels in brain and spinal cord were higher(approximately 3 μg/g tissue) after oral, compared to parenteral,administration. This could be the result of the liver's partialbreakdown of the polymer into smaller molecular weight fractions duringthe first pass, thus rendering it capable of penetrating the blood-brainbarrier.

In summary, with respect to clinical pharmacokinetic considerations, thedata indicate that ¹⁴ C-labelled acemannan (1) reaches peak blood levelswithin 8 hours or less by all routes studies, (2) has a relatively longbiologic half-like, which would allow therapeutic dosing intervals ofseveral days, and (3) achieves measurable levels in all tissue systemsevaluated, including the central nervous system.

These pharmacokinetic data indicate that acemannan levels in bloodand/or tissue can duplicate those levels known after injection or oraladministration to produce therapeutic antitumor or antiviral effects invitro. For example mice implanted with virally-infected Norman MurineMyxosarcoma (NMM) cells and injected IP within 24 hours with 1 mg/kg ofacemannan showed 35% survival after 60 days compared to 0% survival inNMM-treated control mice (Peng et al., submitted for publication, 1990).Expected peak blood levels at an IP dose of 1 mg/kg would be on theorder of 2 μg/ml×1/20 mg/kg). Acemannan added to cultures ofT-lymphocytes at a concentration of only 0.15 μg/ml (2.6¹⁰⁻⁹ M; 60,000MW) increased the generation of cytotoxic T-cells 230% and increased thefunctional capacity of generated cytotoxic T-cells by 138% to destroytarget cells against which they had been sensitized [Womble et al., Int.J. Immunopharmac. 10(8):967-974 (1988)]. Cytotoxic T-cells are thoughtto be generated against tumor cells like NMM cells.

Blood levels of 4-5 μg/ml obtained after oral administration ofacemannan are also significant, since they correspond to theconcentration of acemannan that gives optimal synergism with Zidovudine®(AZT) in vitro. For example, alone 0.001 μg/ml AZT or 3.2 μg/mlacemannan increased the viability of CEM cells infected withHTLV-III_(RFII) virus by no more than 10% together the protective effectof the antiviral combination exceeded 70%. Similarly, a combination of0.1 μg/ml of AZT and 1 mg/ml acemannan resulted in a protective effectexceeding 80% (Kemp et al. submitted for publication 1990).

Thus, in conclusion, this pharmacokinetic study demonstrates thatacemannan concentrations at least as great as those known to work invitro are attainable in vivo.

EXAMPLE 6 Report of Two Initial Clinical Pilot Studies of Acemannan inHIV-1-Infected Patients

Before the discovery that infection with human immunodeficiency virustype 1 (HIV-1) posed a worldwide health threat, there had been onlylimited development of antiviral drugs. Despite the recognition thatmore than 60% of all illnesses in developing countries are caused bydefined viral diseases, very little progress had been achieved in thisarea. Treatment for the most part consisted of the application ofpalliative measures designed to provide comfort and relief of symptomsrather than to interfere with the replication of viruses. The pandemicof AIDS, because of the total ineffectiveness of palliative orsymptomatic treatment, has resulted in the initiation of unprecedentedresearch into new antiviral compounds targeted to interfere with thereplication cycle of HIV.

In the case of the human immunodeficiency viruses, most attention hasbeen directed towards synthesis and development of2',3'-dideoxynucleoside analogs, a class of antivirals that inhibit thevirus-encoded reverse transcriptase. One of these compounds, AZT,remains the only drug approved for the treatment of AIDS. Unfortunately,numerous studies have demonstrated that the compound is extremely toxicin vivo, and its efficacy, although high in vitro, may be considerablyless so in vivo [Richman et al., N. Engl. J. Med., 317:192-197 (1987)].

Two studies assessed the response of human immunodeficiency virus type 1(HIV-1) infection to acemannan and determined whether laboratory valuescould be used to predict response to treatment. The protocol wassubmitted to the FDA, as an individual physician investigational newdrug exemption and approved by the Institutional Review Board of theDallas-Ft. Worth Medical Center. Subjects who were HIV-1 antibodypositive and symptomatic were treated with approximately 400-800 mg oralacemannan daily and evaluated clinically using modified Walter Reed(MWR) clinical scoring. CD4/CD8 lymphocyte counts and HIV-1(p24) coreantigen levels indicated immune competence and active virus load. In thefirst study, the 15 original subjects had an average MWR of 5.6, butafter 350 days of therapy the surviving 13 had an average of 1.8. CD4levels in ten subjects increased from 346mm³ to 471/mm³ within 90 daysand to 610/mm³ at 180 days. Five of the 15 patients had detectable serumcore antigen; by 350 days only 3 of 13 had detectable, but reduced,serum antigen. Data from this first study suggested that values for CD4and serum antigen levels could predict the response to acemannan. Asecond study with 26 subjects confirmed this. The aggregate group had anaverage MWR of 3.0 at the start, and 90 days later their average was1.8. The CD4 level of 16 "responders" rose from 313/mm³ to 372/mm³during this period, but in 10 others went from 63/mm³ to only 77/mm³.Fifteen of 16 individuals predicted to respond favorably had improvedMWR, increased CD4 counts and reduced antigen, indicating that theextent of immunosuppression and viral load influences response totherapy.

EXAMPLE 7

A Phase II Study of Acemannan Alone and With AZT Among Symptomatic andAsymptomatic HIV Patients

Forty-seven HIV+ patients (23 asymptomatic patients, 24 ARC patients)participated in a double-blind randomized phase II study of acemannan.The protocol was approved by the relevant ethical committees of theHospital St. Pierre in Brussels, Belgium. In order to evaluate safetyand tolerance, with or without concomitant AZT therapy, acemannan wasadministered during 24 weeks at a daily dose of 1000 mg (2 capsules of125 mg, 4 times daily). The 23 asymptomatic patients were blindlyallocated to receive either acemannan (11 patients, group 1) or placebo(12 patients, group 2). Of the 24 ARC patients who received 1000 mg AZTdaily during the study, 12 patients (group 3) also received acemannan1000 mg (2 capsules of 125 mg, 4 times daily) and 12 patients (group 4)also received placebo. Thirty-three out of 47 patients (70%) completedthe 24-week study period (respectively 6, 9, 9 and 9 in the 4 groups).Reasons for withdrawals were: clinical evolution (8 patients: 3, 3, 0,2), patients' own will (5 patients: 2, 0, 2, 1), or death (1 patientcommitted suicide in group 3). None of the patients dropped from theprotocol because of side effects or poor tolerance. There was astatistically significant difference in the incidence of adverse drugreactions, mainly nausea, between groups 1 & 2 and 3 & 4, due to AZTtherapy. No difference occurred between acemannan groups (1 & 3) andplacebo groups (2 & 4). Hematological data were statistically comparableamong 4 groups at study entry. At week 24 there were statisticallysignificant differences for red blood with or without AZT but nodifference between placebo and acemannan patients. There was no liver orrenal toxicity among the 4 groups.

The 12 patients treated with AZT and acemannan (group 3) showed astatistically significant improvement of Karnofsky score (K) aftertherapy (p<0.008) (mean K=84 at entry, 90 at exit) when compared topatients treated with AZT alone (group 4) (mean K=81 at entry, 83 atexit). There was no statistically significant difference between group 3and 4 regarding occurrence of adverse events although 2 patients treatedwith AZT alone developed AIDS (1 Kaposi, 1 esophageal candidiasis)compared to none under combination therapy. Comparison of CD4 cell countof AZT-treated patients showed a significantly greater improvement(p=0.01) at the end of the study among those treated with combinationtherapy (mean CD4 263/mm3 at entry, 369/mm3 at exit in group 3 comparedto 145/mm3 at entry and 252/mm3 at exit in group 4). We conclude thatacemannan is a very well-tolerated compound with no biological toxicityand that among ARC patients acemannan would have a role as adjunctivetherapy to AZT in the management of HIV infection.

EXAMPLE 8 Concentration-Dependent Inhibition of HIV-1 Replication andPathogenesis by Acemannan In Vitro

Peripheral blood mononuclear (PBM) cells and two defined CD4+ celllines, MT-2 and CEM-SS, were used as target cells for HIV-1 infectionsand treated with various concentrations of acemannan. Viabilities weredetermined either by the trypan blue dye-exclusion test or by metabolicconversion of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazoliumbromide] to formazan by viable cells. Virus replication and load weremeasured by hybridization of cell-associated viral RNA and cell-free RNAwith an HIV-1 probe prepared from the POL gene. Protection of PBM cellsby acemannan treatment was shown to be concentration-dependent. Percentprotection ranged from 14-100% for cells treated with 3.2-100 μg/ml ofacemannan. Protection by acemannan treatment of HIV-1-infected MT-2cells was not only concentration-dependent but also multiplicity ofinfection-(MOI) dependent. Protection of CEM-SS cells infected at anMOI=0.01 and treated with 62.5 μg/ml of acemannan exceeded 85%. Inaddition to an increase in cell viability, a concentration-dependentreduction in syncytium formation was observed. Syncytia could not bedetected in cultures treated with ≧62.5 μg/ml of acemannan. Aconcentration-dependent reduction in virus replication was also observedfor treated PBM cells. Treatment of PBM cells with concentrations ofacemannan ≧62.5 μg/ml resulted in a 95-100% reduction in detectablecell-associated viral RNA. Treatment of virus-infected CEM-SS cells withacemannan concentrations ≧62.5 μg/ml caused >60% reduction in cell-freevirus. Acemannan treatment inhibits virus-induced cell fusion, increasesinfected cell viability, reduces virus load and suppresses productionand/or release of free virus. Cytotoxicity due to acemannan was notobserved at any test concentration.

EXAMPLE 9 Synergistic Antiviral Effects of Acemannan in Combination WithAZT (Zidovudine)

The protective effects of combinations of AZT and acemannan weremeasured in vitro using HIV-1-infected MT-2 cells at a MOI of 0.03.Checkerboard titration of the two drugs indicted that a synergisticprotective effect occurred. Concentrations of acemannan of less than 125μg/ml were most effective in this respect.

It is now clearly recognized that some form of combination chemotherapywill be required in order to increase the efficacy of AZT while limitingits long term toxic effects and circumventing the further development ofresistant HIV strains. For this reason, as well as the obviousbeneficial effects of acemannan on clinical HIV infection whenadministered with AZT, it was decided to determine whether these twocompounds had a synergistic inhibitory effect on HIV replication invitro.

Virus Strains

The HTLV-IIIB strain of HIV-1 was obtained from Dr. R. Gallo, NIH,Bethesda, Md. Viral stocks were prepared by propagating the virus in H9lymphoid cells. A stock preparation of the virus was stored at -80° C.The 50% tissue culture infective dose (TCID50)/ml of cell-free viruspool stock was determined by end-point titration using MT-2 cells.Multiplicity of infection (MOI) was determined by the method of Reed andMuench.

Cell Lines

MT-2 cells were propagated in RPMI-1640 supplemented with 2 mML-glutamine and 15% (v/v) fetal bovine serum. MT-2 cells naturallyexpress CD4 on their surface and are thus good target cells for HIV-1infection. In addition, they rapidly undergo cytolysis at low levels ofvirus replication.

Primary testing of antiviral activity

MT-2 cells were first treated with polybrene (2 mg/ml) for 30 min andthen infected with HIV at a MOI of 0.03. After virus absorption, thecells were pelleted and resuspended in complete medium. The infectedcells were then dispensed (2×10⁴ cells/100 μl/well) into 96-wellmicrotiter plates. Each drug was diluted in medium from a stock solutionof 2 mg/ml in six serial half log₁₀ dilutions. AZT was tested from thehighest concentration of 10 μg/ml to a low concentration of 0.032 μg/ml.Acemannan was tested at 500 μg/ml and diluted down to a lowconcentration of 15.62 μg/ml. Parallel assays were performed intriplicate, and drug cytotoxicity was measured at parallelconcentrations in duplicate. Controls included uninfected, untreatedcell cultures and virus-infected, untreated cultures. Plates wereincubated for 7 days in a humidified atmosphere of 5% CO₂ in air. On day7 post-infection, cell viability was measured by the addition of MTT(450 μg/ml) to the test plates. A solution of 10% sodium dodecyl sulfatein 0.01N HCl was then added to dissolve the MTT formazan that wasproduced. The color intensity is a function of the amount of formazanproduced which, in turn, is proportional to the number of viable cellsin each well. Plates were read at a wavelength of 570 nm on a Vmax platereader (Molecular Devices, Inc.). The percent change in cell viabilitywas calculated using the following formula: ##EQU3## where: TI is theoptical density (OD) in treated, infected cells,

TO is the OD in treated, uninfected cells,

OI is the OD in untreated, infected cells, and

OO is the OD in untreated, uninfected cells,

Antiviral activity of acemannan-AZT combinations

Antiviral activity of acemannan in combination with AZT at variousconcentrations was evaluated using the microtiter infection assaydescribed above. For each mixture, defined amounts of the test compoundswere dissolved in RPMI-1640 , and 0.1 ml of each dilution was added totest wells. Combinations were evaluated in duplicate, and treateduninfected controls were used to determine drug cytotoxicity. Eachcompound was also evaluated alone at non-cytotoxic concentrations. ThusAZT was tested at concentrations ranging from 0.32 to 10 μg/ml andacemannan was evaluated at concentrations ranging from 15.62 to 500μg/ml. The percent change in cell viability was determined as describedabove.

The effect of AZT-acemannan combinations on HIV-infected MT-2 cellviability is shown in FIGS. 1 and 2.

Acemannan alone conferred a maximum of 40% protection at a dose of 60μg/ml. At higher doses the protective effect was erratic. AZT aloneconferred a maximum of 60-67% protection at doses ranging from 0.03 to0.32 μg/ml. Cytopathic effects could, however, be completely abolishedby 0.32 μg/ml AZT in the presence of 15.62 to 250 μg/ml acemannan.

The data presented here clearly show that AZT and acemannan exercise asynergistic protective effect on MT-2 cells infected in vitro withHIV-1. These results support the clinical evidence for the same effect.Synergism, by definition, implies that the effect of the mixture ofcompounds is greater than the sum of their effects when acting alone.Thus when 0.01 μg/ml of AZT was mixed with 15.62 μg/ml acemannan(neither of which was protective on its own), 32% cell viability wasachieved. Indeed, at unprotective acemannan concentrations of 31.25 and15.62 μg/ml, significant enhancement in the protective effect of 0.1μg/ml AZT was seen.

When 0.32 μg/ml AZT (65% protection) was mixed with 62.5 μg/ml acemannan(40% protection), the mixture gave 100% protection. Obviously, in thiscase any synergistic effect would be hidden because it is not possibleto detect a viability over 100% and the effect gives a spuriousappearance of additivity.

The appearance of this synergistic effect implies that AZT and acemannaninterfere with viral replication at different stages of its cycle. As aresult, a sublethal hit at one stage in the HIV replication cycle maycomplement a hit at another stage. The combination of two hitscollectively exerts a lethal effect on the virus. AZT, of course, iswell recognized as an inhibitor of the reverse transcriptase of thevirus. Acemannan, on the other hand, possibly interferes withglycosylation and HIV envelope processing.

EXAMPLE 10 Acemannan Used in Treating Cutaneous Ulcers

An 83-year-old female patient, TB, developed an ulcer, 25 mm indiameter, on the lateral margin of her left foot. The ulcer had beenpresent for several months and had failed to respond to severaltreatment regimens.

The wound was treated with the product of Example 3 of U.S. Pat. No.4,735,935 and the product of Example 7 of U.S. Pat. No. 4,735,935 usinga three-times-daily treatment schedule. The clean wound was soaked for15 minutes with the product of Example 2 of U.S. Pat. No. 4,735,935.Excessive product was absorbed from the wound with a dry, sterile 4×4gauze. The product of Example 7 of U.S. Pat. No. 4,735,935 was thenapplied in a quantity sufficient to cover the wound and to prevent wounddehydration between dressing changes.

The progression of wound healing was measured by interval photographsand planimetry of the wound defect. The progression of wound closure isshown in Table 11.

                  TABLE 11                                                        ______________________________________                                        PROGRESSION OF WOUND HEALING                                                              Wound Area Percentage                                             Day         (Sq. In.)  of Healing                                             ______________________________________                                         1          1.24       0.00                                                   28          0.51       58.87                                                  77          0.29       76.61                                                  83          0.12       90.32                                                  97          0.00       100.00                                                 ______________________________________                                    

The epidermal defect was substantially closed in 12 weeks; completeclosure occurred in 14 weeks.

EXAMPLE 11 Acemannan Used as a Treatment For Tic Douloureux

Tic douloureux, or neuralgia of the fifth cranial nerve, ischaracterized by attacks of severe, unbearable pain over one or morebranches of the trigeminal nerve. The pain usually is transient, andattacks may be precipitated by touching some area of the face--theso-called trigger zone.

The cause and cure of this painful disease are unknown. Several attemptsto treat the disorder have met with little or no success. Varioustreatments have included analgesics, phenytoin, peripheral avulsion ofthe involved nerve branch as it leaves the skull, and injection of 98%alcohol into the gasserian ganglion.

A more drastic treatment--sectioning the sensory root of the nerveproximal to the ganglion--leaves the patient permanently withoutsensation in the area supplied by the sectioned nerve. Another recenttreatment attempt uses carbamazepine and phenoliophendylate injections.However, these injections can be complicated by unpleasant numbness andserious side effects.

None of the previously cited treatments is desirable.

A 43-year-old woman was diagnosed as having tic douloureux. The affectedarea included the first and third divisions of the trigeminal nerve onthe right side.

The patient could trigger the pain by brushing or combing her hair onthe right side. She had been treated unsuccessfully with diazepam(Valium), antihistamines, analgesics, propranolol hydrochloride(Inderal) and phenobarbital. The patient said she had not had apain-free day since the onset of the disease.

The proposed therapy involved drinking 1 to 2 oz. of the product ofExample 2 U.S. Pat. No. 4,735,935 daily for 3 months. After that period,the therapy was evaluated.

The patient's pain diminished significantly within 2 weeks of initiatingtherapy. She said she felt well for a few weeks. However, she then wenton a 2-week trip during which she did not drink the product, andsymptoms and pain returned. After she resumed the medication, however,the pain disappeared within a few days. For the next few weeks, sheagain felt well.

After drinking the product daily for more than 6 months withoutinterruption, she reports that she can brush and comb her hair withouttriggering the pain. Her appearance has improved, and she says she feelsbetter than ever before.

EXAMPLE 12 An Exploratory Clinical Pilot Study Utilizing Acemannan inInflammatory Bowel Disease

Inflammatory bowel disease (IBD) is a collective term for Crohn'sdisease, ulcerative colitis and other conditions of the gastointestinaltract. Crohn's occurs mainly in the ileum and colon, whereas ulcerativecolitis is limited to the colon. At least three credible hypotheses havebeen set forth to explain the etiology of IBD. One holds that an unknowninfectious agent, such as a slowly growing bacterium or virus, triggersthe immune system and sets up a chronic inflammatory response. Thesecond holds that this same sequence of events is caused by a toxicsubstance, such as food-borne or environmental contaminants. The thirdhypothesis suggests that the inflammatory response is an autoimmunecondition. However, the precise cause(s) of the disease remains unknown.

A. Patient Selection

This study protocol was submitted to the FDA as an individual physicianinvestigational new drug exemption and approved by the InstitutionalReview Board of the Dallas-Ft. Worth Medical Center. Patients wereselected without regard to age, sex, racial or ethnic background, andall patients were volunteers. Each received an informed consent briefingby the physician, and each was required to sign an informed consentform.

Only patients with a combination of the following symptoms and signs ofIBD were admitted:

1. Diarrhea (number of bowel movements)

2. Blood in stool (occult blood)

3. Excess mucus production

4. Spontaneous abdominal pain

5. Abdominal pain on palpation

6. Constant cramping

7. Other (weight loss, etc.)

The above symptoms were used to arrive at a clinical evaluation score of0 to 7 with 1 indicating a single symptom and 7 indicating all symptomswere present. A score of 0 indicated the patient was asymptomatic.

B. Endoscopic Evaluation.

Endoscopy was utilized to score patients pre- and post-therapy accordingto the following criteria:

1. Ulcerations

Confluent

Spotty

Linear

Segmental

2. Hyperemia

3. Exudate

4. Other

The same experienced endoscopist scored listed mucosal appearancesaccording to distribution and severity. A score of 0 indicated thepatient had normal appearing mucosa 5 denoted most severe.

C. Histological Evaluations.

Scoring of histolocial findings were recorded as follows:

Exudate, Ulcerated mucosa, Edema, Plasma cells, Lympohocytes,

Polymorphonuclear cells, Eosinophils, Granulomas, Crypt abscess,

Fibrosis, Other

The above clinical, endoscopic and histopathlogical criteria were usedto grade manifestations of IBD and to quantify response to acemannantreatment. Physical examinations with endoscopy and histologicalsampling were limited to regularly scheduled visits. Patients werepermitted to withdraw at any time without cause and without impact upontheir usual therapy. Acemannan was furnished by Carrington Laboratories,Inc.

D. Clinical Results.

Nine IBD patients were admitted and were treated daily with 200 mgacemannan in capsules. Patients ranged in age from 14 to 46 years andincluded four females and five males. Typically, the patients hadabdominal pain, diarrhea or multiple bowel movements; the stools wereusually bloody and watery with an increase in mucus production or acombination of these elements. Initial endoscopic examination revealed aspectrum of mucosal alterations ranging from vascular congestion withmucosal friability to focal, extensive and confluent ulcerations, termed"pan-colitis." Histological Histological examination of bowel biopsiesrevealed damage ranging from a non-specific increase in chronicinflammatory cells to frank ulceration with numerous polymorphonuclearcells and eosinophils. All patients were presented as nonresponsive toconventional agents, including one or more of the following: Azulfidine,prednisone, 6-mercaptopurine and Flagyl. Imodium and tranquilizers wereoften added to the above agents.

The response to acemannan medication was uniformly favorable, with allscores improving in all patients. The average pre-and post-medicationscores were as follows:

    ______________________________________                                        Average pre-treatment clinical score                                                                  4.56 (average of                                                              nine patients)                                        Average post-treatment clinical score                                                                 0.44 (average of                                                              nine patients)                                        Average pre-treatment endoscopic score                                                                3.88 (average                                                                 of eight patients)                                    Average post-treatment endoscopic score                                                               0.00 (average of                                                              two patients)                                         Average pre-treatment histological score                                                              6.25 (average                                                                 of eight patients)                                    Average post-treatment histological score                                                             N/A (Patients                                                                 all refused biopsy)                                   ______________________________________                                    

No adverse effects attributable to acemannan were observed at any timeduring the study. Some patients who were quite experienced with theirown disease expression reported they were virtually free of pain andsymptoms within 2-5 days. In others, particularly those with focalsegmental disease (Crohn's and ileitis), the effects of acemannan wereslower and less dramatic. All patients refused the post-treatmentbiopsy, and only two patients accepted post-treatment endoscopy. Thefollowing reasons were given by the patients: (1) these procedures areuncomfortable, and (2) the cost was not justified because of theirimproved condition.

Two patients were episodic in their intake of acemannan, taking it onlywhen symptomatic. Both reported relief of symptoms in 24 to 48 hoursafter consuming the medication; however, mild symptoms returned in 4-6weeks after discontinuance of acemannan treatment. Subsequently, 2-3days of acemannan treatment again relieved symptoms. Acemannan provideddramatic clinical improvement in the acute inflammatory phase of thedisease.

EXAMPLE 13 The In Vitro Effects of Acemannan on Measles Virus

Measles virus was incubated with various concentrations of acemannan andthen added to susceptible cultures of VERO cells. The purpose of thisexperiment was to determine whether acemannan would inhibit infection orinactivate measles virus treated with acemannan prior to introductioninto a susceptible cell culture. Acemannan-treated virus did not infectthe VERO monolayer as evidenced by the absence of cytopathic effects(CPE) of the virus at a threshold concentration of 2.5 mg/ml. Completeabsence of CPE was achieved at 5 mg/ml of acemannan in the virusinoculum.

African Green Monkey kidney cells (VERO cells) were used as the targetcells. Measles virus was titrated to obtain a plaque count of 30-50plaques/ml (20 TCID units/0.05 ml) on the virus/cell monolayer.Acemannan at different concentrations was then introduced into mediacontaining this fixed amount of virus.

The concentrations of acemannan were made in complete tissue culturemedium. An aliquot of rubella attenuated virus vaccine was used for eachtitration. The mixtures were pre-incubated at 30° C. for one-half hourand added to previously prepared VERO monolayer in tissue culturechambers.

The results of combining measles virus with various concentrations ofacemannan incubated five full days on confluent VERO cell monolayers areprovided in Table 12.

Repetitious challenges with various concentrations of acemannan showedthat a protective concentration was achieved between 2 mg/ml and 4mg/ml, this being a transition zone for inhibiting measles virusinfectivity. Note Table 12. It is apparent that the 5 mg/ml acemannanlevel consistently provided protection to the VERO cell monolayerchallenged with measles virus pretreated with acemannan.

In this pilot study the effect of acemannan on measles virus wasevaluated by comparing 1) VERO cells only (negative controls, 2) VEROcells inoculated with measles virus (positive controls), and 3) VEROcells inoculated with measles virus that had been pre-treated withacemannan. A significant reduction in plaque formation occurred in theacemannan-pretreated virus-infected cultures (#3) as determined byplaque count assay. Complete protection of cultures from virus infectionwas achieved when virus was pretreated with 5 mg/ml acemannan.

                                      TABLE 12                                    __________________________________________________________________________    EFFECT OF ACEMANNAN CONCENTRATION                                                       VIRUS    #                                                          DATE  DIL DOSE     1   2   3 4 AV. INF.                                       __________________________________________________________________________    09/10/86                                                                            5   25   34  1(?)        1   0                                                    2.5      12.530      0   0 0                                                  1.25     6.2516      1   1 6.25                                               0.625    3.12512     4   4 3.33                                     09/17/86                                                                            5   20   100+                                                                              0   0       0     0                                              2.5          20  30      25    25                                             1.25         60  30      50    50                                             0.625        100+                                                                              100-    100+  100                                            0.3125       100+                                                                              100+    100+  100                                            0.1525       100+                                                                              100+    100+  100                                                                     I                                              10/08/86                                                                            5.0 20   100+                                                                              0   1       1     1                                              4.5          1   0       1     1                                              4.0          0   2       2                                                    3.5          10  1       5.5   6                                              3.0          9   0       4.5   5                                              2.5          5   9       7     7                                              2.3 10       0           0     0                                              1.0 5        0           0     0                                        10/12/86                                                                            5.0 20   5.5 0   0   0 0 0     0                                              4.5          0   0   0 0 0     0                                              4.0          0   0   0 0 0     0                                              3.5          1   0   0 0 0.25  4.5                                            3.0          1   0   0 0 0.25  4.5                                            2.5          0   1   1 1 0.75  11                                             2.5 12.5     0   0   0 0 0     0                                              1.0 6.25     0   0   0 0 0     0                                        10/01/86                                                                            5.0 20   6.0 0   0       0     0                                              4.5          0   0       0     0                                              4.0          0   0       0     0                                              3.5          0   2       1     16.6                                           3.0          1   2       1.5   25                                             2.5          3   3       3     50                                             2.5 10       0   0       0     0                                              1.0 5        0   0       0     0                                        __________________________________________________________________________

EXAMPLE 14 Ability of Acemannan to Reverse Measles Virus Infection InVero Cell Culture

VERO cells were incubated with medium containing 40 TCID/ml of measlesvirus for various periods of time (0.5 to 6 hours) prior to the additionof 5 mg/ml of acemannan. Incubation with acemannan after cells wereexposed to the measles virus did not protect the VERO cells frominfection.

VERO cells were incubated for 0.5 to 6 hours with medium containing 40TCID/ml of measles virus. The VERO cells were then washed with freshmedium to remove any unbound virus. Medium containing 5 mg/ml acemannanwas then added to the cultures, and the cultures were examined forcytopathology after five days.

Results of this experiment are shown in Table 13:

                                      TABLE 13                                    __________________________________________________________________________    EFFECT OF BRIEF INCUBATION OF VERO CELLS WITH                                 MEASLES VIRUS FOLLOWED BY ACEMANNAN                                           TREATMENT                                                                              VIRUS    #                                                           DATE  DIL                                                                              DOSE     1  2  3  4  AV.  INF.                                       __________________________________________________________________________    09/29/86                                                                            5.0                                                                              OT 20 L/0.5 mL                                                                         25 25 25 10 21.25                                                 5.0                                                                              0.5 hr    1  3 10  2 3.5  100                                              5.0                                                                              1.0 hr    1 10  9 16 9    16                                               5.0                                                                              4.0 hr    8 21 25  7 15.25                                                                              42                                               5.0                                                                              6.0 hr    x 18 15  4 12.3 71                                                                            58                                         11/14/86                                                                            5.0                                                                              OT 20 L/0.5 mL                                                                         13 17 17 25 18                                                       0.5 hr                    100                                                 1.0 hr                                                                        4.0 hr                                                                        6.0 hr                                                               06/10/87                                                                            5.0                                                                              OT 20 L/0.5 mL                                                                         100                                                                              100   100                                                                              100                                                   5.0                                                                              0.5 hr    8  8 10   9                                                                              8.75 14                                               5.0                                                                              1.0 hr   10  8  9 11 9.5  15.5                                             5.0                                                                              4.0 hr   25 15 25 30 23.75                                                                              38                                               5.0                                                                              6.0 hr   24 24 25 31 26   42                                         __________________________________________________________________________     Average of two assays for graph 0t = 100                                      0.5 hr = 15                                                                   1.0 hr = 28.8                                                                 4.0 = 54.5                                                                    6.0 hr = 50                                                              

A lower infection rate was noted in the half-hour and 1-hour acemannanpre-incubation cultures. No clinically significant protection of VEROcells was noted in cultures pre-incubated for longer periods withacemannan.

VERO cells pre-incubated wit measles virus were not significantlyprotected from infection by addition of 5 mg/ml of acemannan after theinfection period had ended.

EXAMPLE 15 Project to Determine The Effectiveness of Acemannan on TheInduction of a Protective Immune Response in Commercial Poultry

Nationally, losses from disease and management related problems cost thepoultry industry in excess of $2 billion annually. Infectious agentssuch as infectious bursal disease virus (IBDV), a retrovirus thatinduces mortality and/or morbidity associated with immunosuppression,cause severe economic losses to the poultry industry. IBDV specificallytargets precursor B-cells in the bursa of Fabricius leading to selectivedestruction of the humoral arm of the immune system. This causes animmunosuppressed state akin to Acquired Immune Deficiency Syndrome(AIDS).

The poultry industry routinely vaccinates flocks against IBDV by oraladministration of live virus or by subcutaneous injection of inactivatedvirus. Although both methods of vaccination may effectively elicit animmune response, inherent problems associated with the use of vaccinesare introduced. Live virus vaccines are more effective in theelicitation of a protective immune response to a specific strain, butthe virus itself may revert to virulence, or replication of the vaccinestrain may cause transient immunosuppression leading to increasedsusceptiblity of the flock to secondary pathogens. Killed virus vaccinesdo not have the same problems as those associated with live virusvaccines, but immune responsiveness is diminished and is dose-dependent.Numerous alternatives to vaccination that involve complicated high-techsolutions are being evaluated but directed modulation of the immuneresponse by inclusion of an additional component in a killed-virusvaccine represents a potentially simple solution.

Acemannan, on the basis of preliminary observations, acts as animmunomodulator, and this project was designed to determine whether thiscompound stimulates the immune response to a killed infectious IBDVvaccine.

A. Animals

Chicks hatched from eggs purchased from SPAFAS, Inc. were used for allexperiments. Eggs were hatched, and day-old chicks were placed inHorsfall Units.

B. Antigen

Bursa Vac K (oil emulsion)--acemannan used: Lot #80226-001; resuspendedat 1 or 2 mg/ml (see experimental design).

C. Experimental Design

Study #1 (Group 1).

For Study #1, 25 2-week-old chicks were divided into five groups. Thechicks in each group were vaccinated as follows:

Group 1--control, sham inoculated

Group 2--inoculated subcutaneously over the back with 0.5 ml of oilemulsion vaccine

Group 3--inoculated subcutaneously with 0.25 ml of oil emulsion vaccine(Bio-Burs K; Key Vet., Gainesville, Ga.) mixed with the 0.25 ml ofacemannan (0.5 mg/ml) suspended in water (1:1)

Group 4--inoculated orally with 0.5 ml of microcapsules suspended inacidic water

Group 5--inoculated orally with 0.5 ml of microcapsules suspended inacidic water with 0.5 mg of acemannan

Study #2 (Group 2).

For Study #2, 117 1-week-old SPF chicks were divided into six groups.The chicks in each group were vaccinated as follows:

Group 1--control, sham inoculated

Group 2--inoculated subcutaneously over the back with 0.5 ml ofacemannan (2 mg/ml) suspended in water

Group 3--inoculated subcutaneously over the back with 0.5 ml of oilemulsion vaccine (Bio-Burs K; Key Vet., Gainesville, Ga.)

Group 4--inoculated subcutaneously over the back with 0.25 ml of oilemulsion vaccine mixed with 0.25 ml of acemannan (1 mg/ml) suspended inwater (1:1)

Group 5--inoculated subcutaneously over the back with 0.25 ml of oilemulsion vaccine mixed with 0.25 ml of acemannan (2 mg/ml) suspended inwater (1:1)

Group 6--inoculated subcutaneously over the back with 0.5 ml of oilemulsion vaccine and over the femoral region with 0.5 ml of acemannan (2mg/ml) suspended in water

For both studies was collected from each chick at weekly intervals, andserum IBDV ELISA titers were determined using commercially availableAgriTech IBDV ELISA kits. FlockChek software, a program marketed byAgriTech Inc., was also used in determining titers.

D. Results

Chicks exhibited no discomfort or side effects as a result ofsubcutaneous or peroral administration of acemannan suspended in wateror oil emulsion.

For Study #1 (Group 1) mean ELISA titers are presented through the sixthweek following vaccination in Table 14:

                  TABLE 14                                                        ______________________________________                                        IMMUNOSTIMULATORY EFFECTS OF ACEMANNAN:                                       STUDY #1                                                                                  IBDV ELISA TITERS                                                 Present     DAYS POST-VACCINATION                                             Group  Antigen  0     7    14   21   28    35   42                            ______________________________________                                        #1     Cont     0     0    0    0    7     107  191                           #2     Em       0     0    54   372  556   218  4983                          #3     Em & Ca  0     5    231  1142 2276  4508 3101                          #4     Mic      0     0    0    2    5     61   127                           #5     Mic & Ca 0     0    1    0    13    150  0                             ______________________________________                                    

Two weeks after primary vaccinations, titers to IBDV started to rise inchicks treated with oil emulsion or oil emulsion supplemented withacemannan. Chicks treated with the oil emulsion vaccine supplementedwith acemannan had an overall mean titer approximately 3.9 times higherthan those vaccinated with oil emulsion vaccine. Three weeks aftervaccination the chicks were revaccinated, with each chick receiving thesame antigen mixture presented in the primary vaccination. One weekafter secondary vaccination, the difference in mean titer ratio hadincreased to approximately 4.1. Two weeks after the secondary injection,when mean titers for both groups had reached their peak, the ratio feelto approximately 2.1. By 3 weeks after secondary vaccination, meantiters for both vaccinated groups had begun to decrease, but thedecrease in titer for chicks vaccinated with oil emulsion alone was moreprecipitous, with a drop in titer of 55% as compared to 31% for chicksvaccinated with oil emulsion supplemented with acemannan. Maintenance ofthe higher titer in birds treated with oil emulsion supplemented withacemannan appears due to prolonged immunostimulatory actions ofacemannan.

Three weeks after the secondary vaccination, chicks from the oilemulsion vaccine group (#2) and the oil emulsion vaccine supplementedwith acemannan group (#3) were redivided into two groups (A and B).Group A chicks were challenged with the homologous live vaccine strain,and Group B chicks were challenged with a virulent field strain. Threedays after challenge, all chicks were necropsied. There was no effect onthe immune system in Group A chicks challenged with the vaccine strain.But all Group B chicks had lesions as demonstrated by histopathology.These were the expected results, but if chicks given only a primaryvaccination had been challenged, it is likely that a greaterpreponderance of lesions in chicks given only the oil emulsion vaccinewould have been seen. If the chicks had been vaccinated with the livevirus vaccine, lesions in the lymphoid organs would have been seen inchicks resistant to homologous virus challenge.

For Study #2, group sizes and the vaccination protocols were changed. Asmay be seen from Table 15, results were inconsistent:

                  TABLE 15                                                        ______________________________________                                        IMMUNOSTIMULATORY EFFECTS OF ACEMANNAN:                                       STUDY #2                                                                                       IBDV ELISA TITERS                                            Present          DAYS POST-VACCINATION                                        Group Antigen        0      7    14    Std. Dev.                              ______________________________________                                        #1    Cont            0     11    1    S.D.  0                                #2    Ca (0.5 mg)    11     37    1    S.D.  0                                #3    Em             21     11   181   S.D. 571                               #4    Em & 0.25 mg Ca                                                                              46      0    5    S.D.  11                               #5    Em & 0.5 mg Ca 188     0   279   S.D. 824                               #6    Em Rt & 0.5 mg Ca Lt                                                                         36     79   504   S.D. 842                               ______________________________________                                    

Differences in the birds were initially noticed two weeks afterinjection. There were more runts than would be expected, and some of thesites where the chicks were banded appeared to be infected; they hadpressure necrosis, which would result in toxin release, in addition tosecondary bacterial infection. In an effort to circumvent the latterproblem the chicks were rebanded and treated with a topical antibiotic.However, the problems described would probably cause overallimmunosuppression, thus voiding the results of this study. Therefore,the experiment was terminated.

In spite of the negative factors associated with Study #2, acemannancaused an overall stimulatory effect of the immune system, i.e., as anenhanced immune response to test antigens administered at sites remotefrom the site of acemannan administration. Although the initialimpression was that acemannan had to be mixed with the oil emulsionvaccine, it appears that an enhanced immune response was elicited whenthe antigen and acemannan were presented separately as well. This resultallows for exploration of alternative vaccination methodologies andapplications for this compound.

Acemannan has adjuvant properties. It increases the persistence oreffective presentation of IBDV antigen within the body, possibly leadingto release of lymphokines and an enhanced lymphocyte response.

EXAMPLE 16 Acemannan Used For The Treatment For Malabsorption Syndrome

Malabsorption syndromes in man cause a wasting condition that caneventually result in death. Specific human syndromes such as sprue andceliac disease can be ameliorated if certain grains containing complexpolysaccharides and enzyme-inhibiting peptides are withdrawn from thediet. The results of this diet change is to reduce the symptoms.However, a major physiological problem remains for the patient;maturation of small bowel intestinal mucosa is arrested due toinhibition in synthesis of glycoproteins essential for cell maturation.This failure of small bowel interaction reduces absorption surface andfurther results in failure to absorb essential amino acids, fatty acids,minerals, vitamins and other critical molecular substances present inthe diet.

A 56-year-old male who had lose over 40 pounds from chronicgluten-sensitive sprue had a rapid reduction in chronic diarrheafollowed by progressive weight gain after taking oral acemannanestimated at 500 to 800 mg/day.

Mannose is required for glycoprotein synthesis. Providing additionalmannose in a diet predictably shifts the velocity of K_(m), increasingthe rate of glycoprotein synthesis. Enzyme synthesis is promoted by theavailability of the critical mannose substrate by mannose-metabolizingenzymes. This increase in glycoprotein synthesis and availabilityresults in small intestine mucosal cell maturation and reduction insymptoms associated with sprue and celiac disease. In addition, thisthermodynamic shift in glycoprotein synthesis has applications to othercategories of disease for which no effective existing therapy exists.

EXAMPLE 17 Acemannan as a Treatment For The Symptoms Associated WithMultiple Sclerosis

Multiple sclerosis (MS) is a neurological disease of unknown etiologyand no effective treatment. Analysis patient data and demographicsindicate the disease is most likely initiated by an infectious agent,probably of viral origin. Analysis of central nervous system lesions,spinal fluid and serum suggests that an autoimmune component is alsopresent. This autoimmune response results in myelin sheath degradation.

A 36-year-old patient who had suffered from multiple sclerosis for morethan 6 years and had been bedridden for 4 months was treated withsteroids after which walking in the house was possible with the aid of awalker. A wheelchair was needed for travel outside the home. Theattending physician advised the patient that without chemotherapy(including treatment with cytoxan), the patient would return to thebedfast state within 6 months.

The patient elected to discontinue all prescribed therapy and begantaking approximately 500 mg oral acemannan daily. The patient observedno change in the status of the disease until about 10 weeks had elapsedat which time she reported feeling better than at any time in theprevious year. Two weeks later she was able to walk longer distancesusing the walker and in one more month was able to walk with the use ofonly a cane. By the time she was to have been bedridden (as predicted byher physician), she was able to shop several hours at a time using acane or walker.

The patient has maintained this level for more than 10 months whilecontinuing to consume about 500 mg acemannan per day with no otherconcurrent therapy.

In 1986, patient F. G. a 57-year-old female presented with a history ofleg weakness, progressive loss of voice and multiple other neuromuscularsymptoms. At Johns Hopkins Medical School, MS was diagnosed with NMRconfirmation. The patient was started on 800 mg/day oral acemannan andan extensive exercise program.

Six weeks after therapy initiation, she was able to go on an oceancruise with limited physical support. The patient's voice progressivelystrengthened and other symptoms improved. At 6 months she reported thather neurologist indicated that plaques visualized by NMR technique hadregressed. The patient continues to experience remission of the symptomsassociated with MS.

EXAMPLE 18 Acemannan Used as an Antiviral Agent in a Plant

Acemannan was evaluated as an antiviral agent against the LaFrancevirus, a major problem in the mushroom farming industry. The compostused was prepared by a modification of the method of Flegg et al.LaFrance virus-infected Agaricus bisporus M8 span was added to preparedcompose at 3% of dry matter. The spawned trays were covered with plasticand incubated for 14 days at 24° C. Acemannan was added to spawnedcompost in a range of doses from 0.01% to 2% (calculated on a dry weightbasis) and placed into 11"×7" trays at 1.0 lb dry matter/tray. Thematerial was mixed evenly with the compost by adding both components tothe sampling bag and mixing thoroughly. Spawned, treated compost wasturned and overlaid with sphagnum peat moss and incubated for a further7 days at 24° C.; the overlay material was then gently mixed, theplastic replaced, and the trays incubated an additional 10 days. Theplastic was then removed and the trays incubated for an additional 7days at 18° C. in an environmental chamber. Mushrooms were harvested atweekly intervals thereafter for three weeks.

Sporophores were analyzed for double stranded (ds) viral RNA byhomogenization in 20 ml STE (1.0M NaCl; 0.5M Trizma base, pH 8.0; and0.01M EDTA), 20 ml LiCi, and 10 ml 10% SDS. The dsRNA was extracted inphenol and the aqueous phase was passed through a Bio-Rad LC columncontaining Whatman CF-11. The cellulose-bound dsRNA was washed and theneluted with STE. After precipitation with ethanol and resuspension incitrate solution, an aliquot was characterized by agarose gelelectrophoresis. The dsRNA patterns were visualized by ethidium bromidestaining. A reduction in dsRNA was shown in the sporophore analyzed fromexperimental trays containing 0.01% to 1.0% acemannan as compared tountreated controls.

EXAMPLE 19 Acemannan Used as a Treatment For Chronic Fatigue Syndrome

Acemannan has been shown to affect chronic viral syndromes in humans. A41-year-old female with a 2 year history of markedly debilitating"chronic fatigue syndrome" (CFS) and elevated Epstein-Barr viral titersreported that taking 800 mg/day of acemannan orally for 6 monthsresulted in complete relief of lethargy. After three excellent monthswithout symptoms, the patient discontinued oral acemannan and there wasa slow return of tiredness with fatigue. Resumption of acemannan rapidlyalleviated the symptoms of the syndrome.

A physician's sister had a prolonged period of chronic fatigue syndromewith elevated Epstein-Barr antibodies. Multiple clinical evaluation andtherapeutic regimens had no effect. The patient started consuming 800 mgacemannan daily and reported a marked improvement followed byelimination of symptoms after 2-3 months of acemannan therapy.

EXAMPLE 20 Combination of Radiation, Chemotherapy and Acemannan For TheTreatment of a Tumor of Embryonic Tissue Origin

Patient W. H. at the age of 41 presented at a medical center with severechest pain. Radiographs suggested a mediastinal mass of possiblevascular origin and the patient was transferred to a cardiovascularcenter. There it was determined that a mass extended from the low neckregion to the diaphragm, grew between the lungs and involved the base ofthe heart. Biopsy disclosed a malignant embryonic sinus tract tumor.Treatment with oral acemannan, (approximately 500 mg/day with radiationand chemotherapy) was instituted. Six years past therapy, the patienthas a normal chest x-ray and lives a normal, active life. A review ofthe literature disclosed 20 such tumors with 100% fatality 9 to 12months post-diagnosis.

EXAMPLE 21 Multi-Drug Combination Chemotherapy and Acemannan Treatmentof a Hepatic Tumor

Patient M. A. presented with the chief complaint of inability to zip uphis pants or get his belt around his abdomen. Computer axial tomographic(CAT) scans in April 1988 revealed multiple tumors in the liver whichextended to the urinary bladder. The liver was largely replaced by over20 tumor masses up to 10 cm in diameter. A life expectancy of 4 to 6weeks was given. The patient had been on 800 mg/day oral acemannan forHIV-1 infection. A multiple chemotherapeutic treatment was initiated andthe acemannan was continued. The patient had minimal side-effects andtoxicity usually associated with cancer chemotherapy. Evaluation at 3months included a CAT scan that revealed an estimated 60% reduction intumor mass. At 6 months, 85% reduction in tumor mass was estimated. Onlyminimal tumor was noted at 12 months, and at 24 months only small scarswith questionable tumor mass remained in the liver. All clinicallaboratory work was normal and the patient is currently doing well.

EXAMPLE 22 Acemannan Treatment of Skin Tumors Associated With HIV

Patient S. G. presented with two black lesions, palpable on his arm thathad been previously diagnosed by biopsy as Kaposi's sarcoma. Acemannangel with 5% DSMO was topically applied to the skin masses on the armwhile similar masses on other parts of the body were not treated.Reexamination at weekly intervals revealed noticeable flattening anddepigmentation of the lesions. Sixty day s after therapy was begun, onlyflat scarred areas remained. Subsequent treatment of other lesions onthe same patient showed the same results.

Patient T. P. D. presented with a palpable ankle lesion withpigmentation of classic Kaposi's sarcoma. The lesion was subcutaneouslyinjected with 1 cc recombinant alpha interferon (Roche). The size andpigmentation were improved by 3 days. Topical acemannan gel on a bandagewas applied and, by the end of 1 week, no evidence of a lesion remained.There was no scarring or alteration of pigmentation.

EXAMPLE 23

Acemannan Treatment of Premalignant Skin Lesions

Subject W. B., a balding physician, had numerous solar keratoses oversun-exposed skin. Two weeks of nightly applications of acemannan gel tothese lesions resulted in removal of the scales, crusts and skinirregularities that were pre-malignant in appearance. This response hasbeen noted in many other mature patients.

EXAMPLE 24

Acemannan Treatment of Edema Associated With Cancer Surgery

In January 1984, patient J. J., a 32-year-old male with unsuccessfulsurgical resection of a pharyngeal primary tumor, radical neckdissection, radiation and chemotherapy was in unmanageable pain due tototal occlusion of the larynx and esophagus. Due to lymphatic blockage,the head was approximately twice normal size and edema had obliteratedall facial features. Life was sustained by a feeding gastroscopy and atracheotomy. Topical acemannan gel (CDWG 0.025%) was applied copiouslyover the entire head, neck and shoulders every 8 hours (TID). By thethird day the edema was noticeably reduced and by the tenth day theexcess tissue fluid was gone. Between the 14th and 16th day theredeveloped fluctuant areas in the neck base, angles of the jaw and behindthe ears. The skin opened and necrotic tissue began to exude from theareas of former hard, infiltrative, nodular tumor. The patient began tocough up masses of gray-white degenerating tumor. Multiple transfusionswere given for the massive hemorrhage which ensured. Slowly, the patientregained the ability to speak, eat soup and breathe through the mouthand nose.

EXAMPLE 25 Combination 5-Fluorouracil and Acemannan Cancer Treatment

Patient C. M. had an abdomino-peritoneal resection of low rectaladenocarcinoma with high mesenteric lymph node metastasis. The patientrefused radiation therapy but elected to accept weekly 5-fluorouracil(5-FU) IV infusions and 800 mg oral acemannan/day. The patient did notsuffer the oral ulcers, severe fatigue or nausea with vomiting usuallyassociated with 5-FU treatment. After an extensive evaluation byisotopic scans and computerized tomography (CAT), the patient had nodetectable adenocarcinoma at 24 months post-surgery and continues to benormal.

EXAMPLE 26 Multi-Drug Combination Chemotherapy and Acemannan CancerTreatment

Patient H. H. had adenocarcinoma of the colon with a resection followedby discovery of rising CEA tumor nodules and CAT scan evidence of aliver nodule. Treatment with oral acemannan (800 mg/day) was begun,along with weekly IV injections of 5-FU (500 mg) dicarbazide (50 mg),and acemannan orally (800 mg). Progressive reduction in tumor size andCEA values occurred with no evidence of side effects or biochemical orhematological toxicity.

In 1990, patient V. G., a 66 -year-old male, presented following totalpneumonectomy with bone and liver metastasis for squamous carcinoma ofbronchiogenic origin. Alkaline and all liver enzymes were elevated morethan triple the upper limits of normal. One month's therapy of 500 mg5-FU and 50 mg dicarbazide IV weekly and 800 mg oral acemannan dailyresulted in reduction of the alkaline phosphatase and liver enzymes tohalf their pre-treatment levels along with improvement of the patient'sgeneral condition.

EXAMPLE 27 Multi-Drug Combination Chemotherapy and Acemannan CancerTreatment With Anti-Male Hormone Therapy

Patient J. R., a 72-year-old male, presented post-surgery andpost-radiation with metastatic adenocarcinoma of the prostrate withrising acid phosphatase (PAP) and prostate specific antigen (PSA). Thepatient was started on oral acemannan (800 mg/day) 5-FU (500 mg) anddicarbazide (50 mg). The rise in tumor markers plateaued, reaching ahigh of 15 units for PAP and 186 U. for PSA. The anti-hormonal agentEulexin was added to the regimen. The PAP dropped within 60 days to 3.0and the PSA to 15. This response was accomplished with no toxicity orside effects at any stage. This patient continues to be monitored whilethe total regimen is continued.

EXAMPLE 28 Acemannan Treatment of Venomous Animal Bites

Acemannan had been shown to alter the body's response to antigen,toxins, allergens and "self" antigens. Two cases of acemannan gel weresent to Swangi Province in Southern China. The Red Cross received theproduct to be used for burns, bed sores, stasis ulcers, and diabeticskin ulcerations. Approximately 1 year later the head of the Red Crosswrote to confirm that acemannan gel had been a useful treatment of theabove conditions; additionally, he reported that it was the besttreatment they had ever used for water snake bite, a common occurrencesin the manually-worked rice paddies. Snake bites often become infectedand are non-responsive to antibiotics; resulting necrosis from profoundischemia in soft tissue causes considerable loss of skin and muscletissue. Occlusive dressing of wounds with acemannan gel eliminated theunmanageable infections and apparently helped restore capillarycirculation to surrounding tissue. This treatment preserved digits,muscles, nerves and soft tissue.

EXAMPLE 29 Acemannan-Treated Cultures of Fibroblasts Revert To NeonatalMorphology and Function

Acemannan-treated cultures of fibroblasts obtained from a 60-year-oldman revealed a change in the morphology of these aging cells. Thischange appeared to evidence a reversal in the aging process in thesehuman cells in vitro. Longer-term fibroblast cultures treated withacemannan (1 mg/ml) in the culture medium resulted in expression ofbiochemical and morphological characteristics of neonatal cells.

In 1989, a physician examined biopsies of facial skin from a surgeryspecimen submitted to the pathology department at Duke University. Thebiopsy was from Mohl's surgery performed for treatment of skin cancer.Acemannan gel was applied post operatively. The specimen exhibited anunusual pattern of necrobiosis with destruction of damaged collagenfibers and rapid regeneration of young collagen fibers by enlargedfibroblasts. The result was rapid remodelling of the structures in thisaged skin specimen.

In 1990, the same physician examined thymuses from dogs given acemannan.These thymuses were 2-6 times larger than the thymuses of the controldogs. Microscopically the thymuses from the treated dogs werehypercellular with evidence of hyperplasia and activation of thymocytes.Increased numbers of T-lymphocytes and "nurse-cell" indicated anapparent expansion in T-lymphocyte clones. The induction of thymusactivity and fibroblast activity in tissues of acemannan-treated animalswould result in the return to function of age-depleted tissues.

EXAMPLE 30 Acemannan Effect on Cholesterol Levels in Animals and Humans

Male dogs given 855 mg/kg/day of acemannan had a statisticallysignificant reduction in serum cholesterol levels which was observedduring a 91-day toxicity study. A similar effect was noted in normalmale volunteers given doses of acemannan ranging from 400 mg to 3200 mgper day. An important effect of acemannan therapy which was observed wasthe statistically significant reduction in serum cholesterol leveltoward normal values for these subjects. Upon entry into the study, themean cholesterol concentration of the 24 subjects was 189 mg/dl; it was174 mg/dl upon exit. Statistical analysis using the "CRUNCH" Softwareversion of the Wilcoxon Signed-Ranks Tests indicated >98% probabilitythat the drop in cholesterol was not due to chance variation.Cholesterol concentrations in seven of the 24 patients decreased morethan 20 mg/dl in 6 days of treatment, and therefore it is difficult toattribute this effect to a simple dietary improvement during thesubjects controlled residence during the study.

EXAMPLE 31 Acemannan Treatment of Injury Resulting From Plants

Two competition hunters who had a supply of acemannan for veterinary usetook it with them on a trip to Africa. After an episode of diarrhea, thehunters decided to take the acemannan themselves. The acemannan wastaken at an 800 mg/day dosage level. The hunters reported that theyexperienced less diarrhea than other hunters. Also, because of briars inthe bush, the hunters suffered numerous cuts on their arms and legs. Thehunters taking acemannan reported that their cuts and scratches seemedto heal virtually overnight. No redness or inflammation developed. Allabrasions were totally healed by the time they flew back to the U.S.Other members of the hunting party suffered serious infections of theircuts and abrasions requiring visits to their physicians for systemic andtopical antibiotic therapy. The two hunters taking acemannan had noinfections and no scarring.

EXAMPLE 32 Acemannan Treatment of Allergies Resulting FromHypersensitivity to Plants

Acemannan was used to ameliorate the inflammatory effect of plantallergens. Subject H. R. M., with a known family history of seasonalhayfever, experienced annual episodes of itching, burning, congestionand watering of mucosal membranes. Starting in 1988, it was found that800 mg/day oral acemannan for 5 days virtually eliminated hayfeversymptoms including sinus headaches produced by the swollen nasal mucosa.In 1989, it was found that acemannan gel applied topically to the mucosaof the eyes and nasal passages at bedtime and every 8 hours thereafterresulted in a similar effect and benefit to H. R. M.

Similar results have been seen with topical administration of acemannanto poison ivy lesions in humans and animals. The severity of and thehealing time for the lesions were significantly reduced.

EXAMPLE 33 Acemannan Treatment of Allergies Resulting FromHypersensitivity to Chemicals

Acemannan was used to ameliorate the inflammatory effect of chemicalallergens. Subject T. R., a professional painter, was on the verge ofquitting his profession due to wheezing and bronchitis induced by vaporsfrom his paints and solvents. After taking 800 mg/day oral acemannan for5 days, his symptoms were relieved. The patient continues to consume 800mg/day of oral acemannan before work each day and is able to continuepainting.

EXAMPLE 34 Acemannan Treatment of Symptoms Associated with Asthma

A 76-year-old male, T. T., with a 72-year history of asthma was taking800 mg acemannan daily for an unrelated condition. After 1 year oftherapy, T. T. told the attending physician he had not used hisaerosolized bronchodilator for over 1 year and a significant amount wasstill in the unit. His wife started that he had averaged 2 units permonth for 15 years and had used a hand aerosolizer for 50 years beforethat for chronic asthma. The patient continues to take 800 mg acemannandaily and no longer has wheezing or chronic bronchitis.

EXAMPLE 35 Acemannan Treatment of Symptoms Associated With CysticFibrosis

A college-age female with 6-month history of cystic fibrosis syndromereported an abrupt return of energy within 2 weeks of instituting oralacemannan therapy at a dose of 800 mg/day.

EXAMPLE 36 Acemannan Treatment of cytomegalovirus Infection as a Resultof HIV Infection

A 27-year-old HIV-1-positive male, M. M., had substernal pain for 2months unrelieved by medications. An esophagoscopy disclosed an erosionof the distal esophageal mucosa. Histopathological staining of biopsiesdisclosed cytomegalovirus organisms in the epithelial cells. Three daysof 1000 mg oral acemannan administered as a lozenge eliminated allsymptoms.

EXAMPLE 37 Acemannan Treatment of Sequela to a Rheumatic Fever Episode

A 25-year-old female, S.M., had the acute onset of arthritis,tendonitis, joint edema, leukocytes, and elevated sedimentation ratefollowing a sore throat caused by an acute episode of post-streptococcalrheumatic fever. The ASO titer was markedly elevated. Her sisters andmother had histories of severe, multiple bouts of acute rheumatic feverwith incapacitation lasting for up to a year.

The patient was administered 800 mg/day oral acemannan. Sedimentationrate and white count were normal by week 6 of therapy and all clinicalsymptoms were gone. The patient returned to a manual labor job requiringhigh dexterity after 8 weeks of therapy.

EXAMPLE 38 Acemannan Used in the Treatment of Autoimmune Disease

A 21-year-old female (E. M.) with a history of pancytopenia, over 2 yearprogressive anemia, leukopenia and thrombocytopenia presented fortreatment in June 1988. She related a history of failure to respond totherapy offered at eight major medical centers in the U.S. Bone marrowtransplant had been offered as the next step in therapy. When firstexamined, the patient had bruising, petechiae, fatigue, low hemoglobin(6.1 gm %) and low platelets (20,000-25,000/mm³) and total white countof 1,500 cells/mm³. Antibody to her own white cells was reported by aspecialty clinical pathology laboratory. The patient was placed on 800mg of oral acemannan for 60 days. Minor improvement in laboratory valueswas noted and her fatigue was minimally reduced. A hematologist wasenlisted to administer low-dose prednisone and horse antithymoctyeglobulin. The former had previously been ineffective. By October 1988the patient's hemoglobin was over 10 gm %, white blood count was 3,500cell/mm³ and platelets were 60,000. By mid-1989, 800 mg/day of oralacemannan was resumed for 6 months. In January 1990 the patient reportedhemoglobin 12.7 gm %, platelets 120,000 and white blood count 5,200cells/mm³. The patient is not on any medication currently and is aprofessional dancer-choreographer in graduate school.

EXAMPLE 39 Acemannan Treatment of Systemic Lupus Erythematosus

A patient with systemic lupus erthematosus was treated by a New Yorkphysician with 800 mg/day of oral acemannan. The physician reported thatall symptoms diminished and laboratory values markedly improved after 8weeks of acemannan therapy. Continued acemannan therapy appeared tocontrol the symptoms associated with this patient's condition.

EXAMPLE 40 Acemannan Treatment of Acute Theumatoid Arthritis

A physician reported that a series of patients with acute rheumatoidarthritis (RA) had elevated RA latex levels and sedimentation rates.After 6 to 8 weeks of 800 mg/day of oral acemannan, the patients hadsignificantly reduced symptoms and markedly improved clinical laboratoryvalues.

EXAMPLE 41 Acemannan Treatment of Chronic Rheumatoid Arthritis

A 65-year-old female, B. L. H., with a 20-year history of chronic RA,deformed joints, subcutaneous nodules and tendinous nodules sufferedwith chronic pain. When the patient was seen in the fall of 1988, shehad been given numerous treatments including gold shots with littlebenefit. Oral acemannan (800 mg/day) was recommended. No benefit wasreported for 6 months. In mid-1989 the patient reported diarrhea ifacemannan was taken more often than twice weekly, but symptoms ofarthritis were improving. In time, 800 mg acemannan three times per weekwas tolerated and symptoms of RA noticeably improved. In January 1990the patient reported that she had experienced her best 6 months of lifein the last 15 years. The effort required and the pain associated withher daily tasks were markedly reduced.

EXAMPLE 42 Acemannan and Antidepressant Therapy of Depression andAnxiety

A psychologist gave 800 mg of oral acemannan per day to patients who hadfailed to respond to psychotherapy and antidepressant drugs taken forsevere depression and anxiety. He reported that by the end of the weekhe could observe that the patients' emotions had become more stable andthat there was a remarkable improvement in attitude for the first timesince the patients had been under long-term observation.

EXAMPLE 43 Acemannan Treatment of Feline Leukemia

Feline leukemia (FeLV) is a retorvirus (class oncovirus) infection inwhich cats present with diverse clinical signs. Infection of thelymphoreticular system is predominant with the majority of animals dyingwithin 3 years. Current treatment for this disease is symptomatic; nocure exists.

Forty-five cats with end-stage FeLV disease were treated byintraperitoneal injection of acemannan weekly for six treatments andobserved for 6 more weeks. The cats were monitored by weeklyexaminations during the treatment period. In addition, laboratory datawere obtained at entry, mid-study and at exit from the study.

Sixty-seven percent of the treatment animals improved during the study.The average survival time of animals not responding to acemannan therapywas less than 28 days.

EXAMPLE 44 Acemannan and Antifungal Drug Treatment of Fungal InfectionsAssociated with HIV

Three HIV-1 patients' records reveal a similar pattern in that thesepatients developed hairy leukoplakia and/or monilial plaques and/orulcers of the oral cavity. Their conditions were usually extensive andpainful. The use of Ketaconazole had improved the condition of somepatients, but others were unresponsive. Within 1 week of adding 800mg/day oral acemannan to their therapy, the patients reportedelimination of these mucocutaneous lesions. Taking acemannan andKetaconazole for 3 to 5 days cleared the outbreak for weeks to months.Continued acemannan administration eliminated or reduced outbreaks ofthe mucocutaneous infections.

EXAMPLE 45 Acemannan and Antioprotozoal Drug Treatment of PneumocystisCarinii Infection Associated with HIV

An HIV-1 study patient receiving 800 mg/day oral acemannan was admittedto an Arkansas veterans hospital for x-ray diagnosis-compatible andsputum proven Pneumocystis carinii pneumonia (PCP). In the experience ofthe V. A. Hospital, HIV-1 patients with PCP take 2 or more weeks torespond to therapeutic measures, if they ever respond. The patientresponded to 1 week of aerosolized pentamidine and continued acemannantherapy. He was dismissed symptom-free.

EXAMPLE 46 Acemannan and Antibiotic Treatment of CryptosporidiosisInfection Associated with HIV

An HIV-1 patient with chronic diarrhea and weight loss was shown to havethe typical acid-fast spores in his stool diagnostic ofcryptosporidiosis. The combination of 800 mg/day oral acemannan and 250mg Q.I.D. ansamycin (rifbutin) daily for 2 weeks rendered the patientfree of diarrhea with resulting weight gain.

EXAMPLE 47 Acemannan and Antitubercular Drug Treatment of ResistantHuman Tuberculosis Associated with HIV

HIV-1 positive patient S. G. sufference progressive weight loss andlow-grade fever for unknown etiology. Discovery of retroperitoneallymphadenopathy and biopsies of the mass eventually revealed a cultureof human tuberculosis. The patient had been on 800 mg/day acemannandaily. Institution of antitubular therapy comprised of 300 mg/dayisoniazid, 600 mg/day rifampin, and 1000 mg/day ethambutol resulted inthe patient's becoming afebrile within 24 hours and gaining weightwithin 10 days.

EXAMPLE 48 Acemannan and Antitubercular Drug Treatment of ResistantAvian Tuberculosis Infection in Humans Associated with HIV

An HIV-1 patient had continuous diarrhea that eventually was shown bysmear and culture to be mycobacterium avian intercellular tuberculosis(MAI). No agent is known to be effective; however, ansamycin wasprovided by C.D.C. In combination, 250 mg Q.I.D. ansamycin and 800mg/day oral acemannan resulted in termination of diarrhea in less than 1week. Within 2 weeks the patient who was losing weight at a rate of 3 to4 pounds per week began to gain weight. Fatigue and general weaknessdecreased. In another HIV-1 patient, C. C., who had biopsy andculture-proven MAI, a similar rapid resolution and negative culturetests were obtained after oral acemannan.

EXAMPLE 49 Acemannan and Multidrug Chemotherapy Including CisplatinCancer Treatment of Breast Cancer

A 38-year old female, J. F., was five years post-mastectomy for ductcell carcinoma of the breast. The patient appeared pregnant due toascites of peritoneal metastasis. CAT scan evidence of liver andmultiple bone sites of lytic tumor were demonstrable. The patient haddelayed chemotherapy and was estimated to be 4 to 6 months from death.The patient was told to start acemannan at an oral dose of 800 mg/dayand to start the recommended multiple agent cytotoxic chemotherapy(adriamycin, cyclophosphamide, mitomysin-C and 5-FU) as offered by herlocal oncologist. She was started on weekly IV administration ofcisplatin in combination with adriamycin and other agents. The patientsuffered no side effects except hair loss and fatigue the day ofinfusion. Subsequent examinations have demonstrated re-ossification ofbone metastasis, elimination of ascites, elimination of palpableabdominal masses, disappearance of the liver mass and weight gain.

EXAMPLE 50 Acemannan Treatment of Cutaneous Fungal Infections

A 40-year-old physician applied acemannan gel to itching, cracked, andburning lesions between and above the bases of his toes. In less than aweek, the doctor reported that the response and healing proved acemannanto be the most effective medication he had used in over 20 years ofperiodic treatment of his chronic athlete's foot. Other patients havereported similar improvement. Remarkably, some patients who were takingacemannan for other conditions reported improvement of athlete's footlesions.

EXAMPLE 51 Acemannan Treatment of Marine Animal Stings

A 40 -year-old female who was scuba diving damaged her knee on firecoral. The lesions covered approximately 9 square centimeters of skinsurface. The normal clinical source of this sting in humans is intenseinflammation for approximately 8 hours followed by delayed healing ofthe skin wound for 14 days. Application of acemannan gel to the woundfor 8 days resulted in complete healing, and scarring was barelydetectable.

SUMMARY

Acemannan has been shown to be effective in treating a number ofconditions where the principal mechanism of resolution or cure requiresintervention by the patient's immune system. Acemannan has directstimulatory effects on the immune system. In addition, acemannandirectly interacts with virus or other infectious organisms, infectedcells, and tumor cells to produce changes in their immunologicallysensitive surface composition to alter the appearance of these agentsand cause them to be recognized by the body's immune system and thendestroyed.

It is thus believed that the operation and administration of the presentinvention will be apparent from the foregoing description. While themethod and techniques shown and described have been characterized asbeing preferred, it will be obvious that various changes andmodifications may be made therein without departing from the spirit andscope of the invention as defined in the following claims.

We claim:
 1. A method for regulating blood cholesterol levels in ananimal, comprising:administering to said animal an amount of polymericacetylated mannan derivative sufficient to reduce serum cholesterol insaid animal.
 2. The method according to claim 1, wherein said polymericmannan derivative is a polydisperse β-(1,4)-linked acetylated mannan. 3.A method for regressing plaques formed in blood vessels of an animal,comprising:administering to said animal an amount of polymericacetylated mannan derivative sufficient to reverse the formation of saidplaques in said blood vessels of said animal.
 4. The method according toclaim 3, wherein said polymeric mannan derivative is a polydisperseβ-(1,4)-linked acetylated mannan.
 5. The method according to claim 1,wherein said animal is human.
 6. The method according to claim 3,wherein said animal is human.